FRET-labeled compounds and uses therefor

ABSTRACT

Reaction mixtures are provided having at least a first nucleotide analog and a second nucleotide analog that produce signals in response to excitation illumination. The signals produced by the analogs have peaks at the same wavelengths, but have distinct signal intensities. The distinct intensities allow for identification of the analogs in nucleic acid sequencing. In some embodiments, FRET-labeled compounds are provided. In certain embodiments, FRET-labeled nucleotide analogs are used, for example, in DNA sequencing or RNA sequencing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/773,519 filed Jan. 27, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/049,541 filed Jul. 30, 2018, now U.S. Pat. No.10,570,445, which is a continuation of U.S. patent application Ser. No.15/379,118 filed Dec. 14, 2016, now U.S. Pat. No. 10,066,258, which is acontinuation of U.S. patent application Ser. No. 13/427,611 filed Mar.22, 2012, now U.S. Pat. No. 9,551,660, which is a continuation of U.S.patent application Ser. No.12/749,859 filed Mar. 30, 2010, now U.S. Pat.No. 8,927,212, which claims the benefit of U.S. Provisional ApplicationNo. 61/164,567, filed Mar. 30, 2009, the disclosure of which areincorporated herein by reference in their entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable.

BACKGROUND OF THE INVENTION

In the analysis of biological processes, researchers are constantlylooking for new and better ways to monitor and analyze both theindividual reactions that make up complex biological systems, as well asobserve the operation of those systems as a whole. In doing so,researchers have developed methods, systems, and compositions thatemploy artificially labeled molecules as model constituents for thosereactions and systems. Observation of the model molecules is facilitatedby the presence of the labeling group. Such labels include radioactivecompounds or radiolabels, chromophores that absorb and/or emit light ofdifferent wavelengths to provide colored indications of an event,chemiluminescent labels that can spontaneously emit light in response toa particular chemical event, fluorescent labels that emit light inresponse to excitation by light of a different wavelength, and reportersystem labels that provide an exogenous, assayable activity or propertyto indicate the presence, absence or change in the model molecule. Suchreporter labels often include exogenous enzymes, binding molecules orthe like that are capable of being identified and even quantified.

In attaching label groups to different model reaction constituents, oneruns the risk that the presence of the label will adversely impact thereaction being observed. For example, large hydrophobic labeling groupscan present issue of steric interference with the progress of thereaction of interest by blocking or not properly interacting with theother reaction constituents. Likewise, labeling components that impactthe chemical properties of the model compound or the reactionenvironment can similarly adversely impact reaction conditions. In othercases, the properties of the label itself may adversely affect thereaction components. For example, the presence of fluorescent moleculesin close proximity to enzymatic reaction components can lead to decay inthe level of enzyme activity through photo-chemically induced reactionintermediates or other impacts.

Further, certain such analyses require the use of multiple labels tomonitor multiple different reaction constituents and/or products. Forexample, in certain sequencing-by-synthesis applications each type ofnucleotide (e.g., A, G, T, and C) is tagged with a different label, anda synthesis reaction is carried out to construct a nascent nucleic acidstrand using a sample nucleic acid as a template. At each position onthe template strand, a nucleotide complementary to the template strandis incorporated into the nascent strand. The newly incorporatednucleotide can be identified by various means, including detection of asignal from a label it carries. The sequence of the template strand isderived from the sequence of complementary nucleotides detected uponincorporation into the nascent strand. Detection of multiple differentlabels in a single analytical reaction adds significant complexity todata analysis, and variability in the performance of the multiple labelscan also adversely affect the ability to “read” the template nucleicacid by virtue of synthesis of the complementary nascent strand.

Accordingly, it would be desirable to provide reaction components thatprovide remedies to some of the issues created by the incorporation oflabeling groups onto components of analytical reactions. The presentinvention provides these and other solutions.

SUMMARY OF THE INVENTION

In certain aspects, the present invention is generally directed tocompounds comprising detectable labels that undergo Förster resonanceenergy transfer (FRET), and these labeled compounds are particularlyuseful in certain analytical reactions. Such detectable labels aretermed “FRET labels” herein, and typically comprise at least twochromophores that engage in FRET such that at least a portion of theenergy absorbed by at least one “donor chromophore” is transferred to atleast one “acceptor chromophore,” which emits at least a portion of thetransferred energy as a detectable signal contributing to an emissionspectrum. In certain preferred embodiments, at least two chromophores ina FRET label emit detectable signals that contribute to a resultingemission spectrum comprising at least two peaks. Such a FRET label canbe termed a “multi-spectral” construct (or a “dual-spectral” constructwhen the emission spectrum has only two peaks). In certain aspects, thechromophores are configured on the compound in order to achieve adesired efficiency of the energy transfer between the donor and acceptorchromophore, where the desired efficiency is chosen to ensure a desiredemission intensity (or range thereof) at one or more emissionwavelengths. In certain preferred embodiments, more than one suchlabeled compound is present in a single analytical reaction, whereineach labeled compound has an emission spectrum that is distinguishablefrom the emission spectrum of every other labeled compound in theanalytical reaction such that the identity of each compound can beunambiguously determined. In preferred embodiments, the emission spectraof certain multiple labeled compounds in an analytical reaction aredistinguishable from one another due to variations in emission intensityat one or more wavelengths as a result of variations in FRET efficiency.In certain embodiments, the multiple different labeled compoundscomprise the same set of chromophores, but have a differentconfiguration and therefore different emission spectra based at least inpart on different FRET efficiencies. In some embodiments,non-FRET-labeled compounds also present in the analytical reactions haveemission spectra that are distinct from the emission spectra of theFRET-labeled compounds of the invention.

In some aspects, the labeled compounds of the invention are analogous tonucleotides and in preferred aspects are readily processed by nucleicacid processing enzymes, such as polymerases. In certain aspects, suchlabeled compounds have incorporation efficiencies that are better thanor at least comparable to triphosphate, tetraphosphate, pentaphosphate,or hexaphosphate analogs.

In certain embodiments, a compound is provided in an analytical reactionthat comprises a label portion comprising a FRET label and a reactantportion, and wherein the FRET label has an emission spectrum comprisingat least two peaks that distinctly identify the reactant portion in theanalytical reaction. In certain preferred embodiments, the FRET labelcomprises at least two fluorophores. The compound may also include alinker portion that maintains a particular orientation of the FRET labelthat ensures a desired FRET efficiency. In certain embodiments, thereactant portion comprises a nucleotide or nucleotide analog, a tRNAanalog, a substrate for an enzyme (e.g., a polymerase), a ligand for areceptor, or an antigen. In some embodiments, a labeled compound isprovided comprising a reactant portion capable of reacting with a firstenzyme, a label portion comprising a FRET label, and a linker portioncoupling the label portion to the reactant portion, wherein the linkerportion maintains a desired conformation of the FRET label, wherein thedesired conformation results in inefficient energy transfer betweenchromophores in the FRET label to produce a distinct and identifiableemission spectrum.

In some embodiments, compositions of the invention include a pluralityof FRET-labeled compounds having optically distinct emission spectra,even in embodiments in which they comprise the same set of chromophores.For example, although two FRET-labeled compounds contain the same two ormore chromophores and emit at the same wavelengths, they are configuredsuch that the emission intensities at those wavelengths are differentand can be used to optically distinguish between the two compounds. Inpreferred embodiments, such optical distinction is due at least in partto differing FRET efficiencies in the two FRET-labeled compounds, whichtypically differ by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%,50%, 60%, 70%, 80%, or 90%. For example, if one FRET-labeled compoundhas a FRET efficiency of 10% and a second FRET-labeled compound has aFRET efficiency of 90%, they differ by 80% FRET efficiency. FRET-labeledcompounds in a single analytical reaction can have FRET labelscomprising some or all of the same chromophores, e.g., fluorophores.Some analytical reactions comprising FRET-labeled compounds alsocomprise labeled compounds that are non-FRET labeled, and suchnon-FRET-labeled compounds can comprise a chromophore (e.g.,fluorophore) present in one of the FRET-labeled compounds. For example,compositions of the invention can include nucleotides and/or nucleotideanalogs coupled to a label portion that does not comprise a FRET label.Compositions of the invention can further include one or more enzymes(e.g., polymerases), receptors, antibodies, molecular complexes (e.g.,ribosomes), or nucleic acids (e.g., RNA, DNA, primers, templates, etc.).Further, methods of the invention can comprise monitoring an analyticalreaction. For example, monitoring of a nucleic acid synthesis reactioncan comprise contacting a polymerase/template/primer complex with such aFRET-labeled compound and detecting a characteristic signal from thelabel portion indicative of incorporation of the nucleotide ornucleotide analog into a primer extension reaction, preferably in realtime, e.g., during the incorporation.

Further, methods are provided for making various compounds comprising aFRET label that include determining a desired FRET efficiency (e.g.,less than 100% of a maximal FRET efficiency for a given combination oflabels), computing a conformation to achieve the desired FRETefficiency, and synthesizing the conformation. For example, a desiredFRET efficiency can be 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95 of the maximum FRETefficiency. Also provided are methods for designing a plurality of FRETlabels, e.g., for use in differentially labeling a plurality ofcompounds in a single analytical reaction. Such methods generallycomprise selecting at least two chromophores for inclusion in theplurality of FRET labels, wherein each can serve as a donor or acceptorfor another, and further determining a set of FRET efficiency values,such that the FRET efficiency value for each of the FRET labels isdifferent from the FRET efficiency value for every other. A distancebetween the chromophores that will achieve each FRET efficiency value iscomputed to generate a set of distances for the plurality of FRETlabels. Finally, a set of linkers is generated that comprises one linkerfor each of the distances in the set, wherein each linker is configuredto separate the chromophores in a single of the plurality of FRET labelsby a single distance in the set of distances. In preferred embodiments,each of the plurality of FRET labels generates a set of emissionintensities at a set of emission wavelengths in an emission spectrumthat is different from that of every other FRET label, e.g., varying theFRET efficiencies by at least about 5%,10%, 15%, 20%, 25%, 30%, 40%,50%, 60%, 70%, 80%, or 90%.

In certain embodiments, a composition is provided comprising a firstcompound and a second compound, both of which comprise a reactantportion and FRET label, wherein the emission spectrum from the firstcompound is distinct from the emission spectrum of the second compound.In some preferred embodiments, the emission spectra from the firstcompound and the second compound both comprise peaks at the samewavelengths, e.g., a first and second wavelength, but wherein theemission intensities at those wavelengths are different between the twocompounds. In some preferred embodiments, a composition of the inventioncomprises a first compound with a first FRET label having a first FRETefficiency and a second compound with a second FRET label having asecond FRET efficiency that is different from the first FRET efficiency,e.g. differing by at least about 5%,10%, 15%, 20%, 25%, 30%, 40%, 50%,60%, 70%, 80%, or 90%. In some embodiments, the two FRET labels share atleast one or more chromophores. In some embodiments, the compositionfurther comprises at least one compound, (e.g., nucleotide or nucleotideanalog) labeled with a single chromophore (e.g., fluorophore), that mayalso be present in at least one FRET label in the composition. In someembodiments, the composition further comprises an enzyme, a nucleic acidtemplate, and/or a primer.

The present invention also provides methods for using the compoundsdescribed herein in performing analytical reactions, such as nucleicacid analyses, and particularly nucleic acid sequence analyses. Incertain embodiments, the analytical reactions of the invention compriseproviding a template nucleic acid complexed with a polymerase enzyme ina template-dependent polymerization reaction to produce a nascentnucleic acid strand, contacting the polymerase and template nucleic acidwith a FRET-labeled compound of the invention, detecting whether or notthe FRET-labeled compound was incorporated into the nascent strandduring the polymerization reaction, and identifying a base in thetemplate strand based upon incorporation of the FRET-labeled compound.In some such reactions, at least one or two of the nucleotide analogs isa FRET-labeled compound and at least one or two of the nucleotideanalogs does not comprise a FRET label, and identification of theincorporated nucleotide analog is based at least in part on a comparisonof the emission spectra generated during polymerization of the nascentstrand. In preferred embodiments, incorporation of labeled nucleotideanalogs, whether FRET-labeled or non-FRET-labeled, is detected inreal-time during nascent strand synthesis. Preferably, the foregoingprocess is carried out so as to permit observation of individualnucleotide incorporation reactions, through the use of, for example, anoptical confinement, that allows observation of an individual polymeraseenzyme, or through the use of a heterogeneous assay system, where labelgroups released from incorporated analogs are detected.

In certain embodiments, methods are provided for distinguishing betweentwo labeled compounds in an analytical reaction, e.g., by labeling thefirst labeled compound with a FRET pair in a first orientation ensuringa first FRET efficiency, labeling the second labeled compound with theFRET pair in a second orientation ensuring a second FRET efficiency,combining the two labeled compounds in an analytical reaction,subjecting the analytical reaction to excitation radiation, detecting asignal emitted from the analytical reaction, and analyzing the signal todetermine a signal FRET efficiency. If the signal FRET efficiency isequal to the first FRET efficiency the signal originated from the firstlabeled compound, and if the signal FRET efficiency is equal to thesecond FRET efficiency the signal originated from the second labeledcompound, and the two labeled compounds are thereby distinguished in theanalytical reaction. Analyzing the signals emitted from an analyticalreaction typically involves determining emission intensities at emissionwavelengths in emission spectra. In certain preferred embodiments, asingle radiation wavelength of the excitation radiation excites both theFRET pair in the first orientation and the FRET pair in the secondorientation. In some embodiments, four labeled compounds aredistinguished from one another by also including in the analyticalreaction a third labeled compound with the first chromophore of the FRETpair (but not the second chromophore) and a fourth labeled compound withthe second chromophore of the FRET pair (but not the first). If thesignal FRET efficiency is not equal to either the first FRET efficiencynor the second FRET efficiency, the signal is further analyzed todetermine if it is characteristic of an emission spectrum of the firstchromophore or the second chromophore, thereby identifying the origin ofthe signal as being the third labeled compound or the fourth labeledcompound, respectively. As such, four different labeled compounds aredistinguishable from one another in an analytical reaction using onlytwo chromophores, e.g., by labeling two of the compounds with a singledifferent chromophore, and by labeling the other two compounds with FRETlabels containing both chromophores in different conformations toprovide detectably different emission spectra.

Further, methods are provided for identification and detection ofindividual labeled reactants in a reaction mixture comprising multipledifferent reactants, where the reactants can be nucleic acids (e.g.,nucleotides or nucleotide analogs, nucleic acid segments, primers,etc.), tRNA analogs, ligands for a receptor, antigens, binding partners,etc. For example, each of the multiple different reactants are labeledwith detectably different chromophore-containing labels, at least two ofwhich are FRET labels that emit at substantially similar wavelengths buta distinctly different emission intensities. The different reactants arecombined in a reaction mixture, and individual labeled reactants aredetected by exposing the reaction mixture to excitation radiation anddetecting an emission spectrum of each of said chromophore-containinglabels. In certain preferred embodiments, a single radiation wavelengthof the excitation radiation excites the FRET pairs in the reactionmixture. In certain preferred embodiments, a first of the FRET labels inthe reaction mixture is configured to ensure a first distance betweenthe constituent chromophores and a second of the FRET labels in thereaction mixture is configured to ensure a distance between theconstituent chromophores that is different than the first distance. Insome embodiments, the two distances are chosen to ensure inefficientenergy transfer resulting in submaximal FRET efficiencies, therebyproducing distinct and multi-peak (e.g., multi-spectral ordual-spectral) emission spectra. For example, the multi-peak emissionspectra typically comprise a peaks resulting from emission from both thedonor and acceptor chromophore in a FRET label. The different FRETlabels in such reaction mixtures typically comprise different linkersthat are structurally different from one another, e.g., that provide fora different spacing or distance between their constituent chromophores.In some embodiments, at least one of the chromophore-containing labelsis not a FRET label, and such non-FRET labels may comprise a chromophorethat is identical to a chromophore in at least one FRET label in thereaction mixture. In some preferred embodiments, the labeled reactantsare nucleotide analogs comprising a single nucleobase.

In further embodiments, methods are provided for determining an identityand relative position of a nucleotide in a template nucleic acidsequence. For example, a template nucleic acid is provided and complexedwith a polymerase enzyme capable of template-dependent synthesis of acomplementary nascent nucleic acid strand. This complex is contactedwith a plurality of differentially labeled compounds, each of whichcomprises an individually detectable label, wherein a subset (e.g., 1-3)of the plurality of differentially labeled compounds comprises anindividually detectable label that undergoes resonance energy transfer(e.g., at a submaximal efficiency, e.g., less than 90%), and a subset(e.g., 1-3) of the plurality of differentially labeled compoundscomprises an individually detectable label that does not undergoresonance energy transfer. The plurality of differentially labeledcompounds further comprise a different base selected from A, T, G, andC, wherein each of the plurality of differentially labeled compoundsthat comprise a given base comprise an identical individually detectablelabel. The reaction is monitored to detect whether any of thedifferentially labeled compounds are incorporated into the nascentnucleic acid strand, where incorporation of one of the differentiallylabeled compounds is indicative of complementarity between a base in thedifferentially labeled compound and a position in the template nucleicacid being processed by the polymerase enzyme. Preferably, the detectionof the incorporated labeled compounds occurs during the incorporationevent, e.g., as the labeled compound is undergoing incorporation. Incertain embodiments, the label portion of the labeled compound is notincorporated, e.g., is removed from the reactant portion during theincorporation event. In certain preferred embodiments, a plurality ofincorporation events is monitored in real time to allow determination ofa sequence of compounds so incorporated, e.g., a sequence of amino acidsincorporated into a nascent polypeptide, or a sequence of nucleotidesincorporated into a nascent polynucleotide. In certain embodiments, asingle reaction being monitored is optically resolvable from otherreactions being simultaneously monitored, e.g., on a single substrate orsolid support. In some embodiments, an array of optically resolvablereaction sites is used to simultaneously monitor individual reactions.

In yet further aspects, methods are provided for distinguishing betweenbinding of multiple reactants. In certain embodiments, such methodscomprise providing a first reactant having a donor FRET chromophore anda second reactant comprising a first acceptor FRET chromophore, whereinthe first acceptor FRET chromophore generates a first emission spectrumduring binding to the first reactant. A third reactant comprising asecond acceptor FRET chromophore is also provided, wherein the secondacceptor FRET chromophore is identical to the first acceptor FRETchromophore, and further wherein the second acceptor FRET chromophoregenerates a second emission spectrum during binding to the firstreactant, wherein the second emission spectrum is detectably distinctfrom the first emission spectrum. A reaction mixture is preparedcomprising the first, second, and third reactants under conditions thatpromote binding of the second and third to the first reactant. Spectralemissions from the first reactant are monitored to detect binding of thesecond and/or third reactant to the first reactant. Detection of thefirst emission spectrum indicates binding of the second reactant to thefirst reactant; detection of the second emission spectrum indicatesbinding of the third reactant to the first reactant. In certainembodiments, at least one of the first and second emission spectra is amulti-spectrum emission spectrum. In certain embodiments, FRETefficiency between the donor FRET chromophore and the first acceptorFRET chromophore is different from FRET efficiency between the donorFRET chromophore and the second acceptor FRET chromophore. Preferably,the detection is performed in real time during the binding events and/orwith single molecule/molecular complex resolution.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 an illustration of an exemplary profile for a set of fouremission spectra according to certain embodiments of the invention.

FIG. 2 illustrates various different linker components for theconstruction of FRET labels as described herein.

FIG. 3 schematically illustrates one embodiment of a system for use withthe compounds and compositions in the methods of the invention.

FIG. 4 provides chemical structures for certain embodiments of FRETlabels described herein.

FIG. 5 provides a synthesis scheme for the Cy5-Cy3 compound depicted inFIG. 4 .

FIG. 6 provides a synthesis scheme for the Cy5-amb-Cy3 compound depictedin FIG. 4 .

FIG. 7 provides a synthesis scheme for the Cy5-amb2-Cy3 compounddepicted in FIG. 4 .

FIG. 8 provides a general synthesis scheme for Cy5-Cy3 compounds.

FIG. 9 illustrates a synthesis scheme for constructing nucleotideanalogs bearing FRET labels.

FIG. 10 provides a synthesis scheme for Cy5-pro6-Cy3-dG6P.

FIG. 11 illustrates individual emission spectra for Cy3 and Cy5.

FIG. 12 illustrates an individual emission spectrum for a Cy5-Cy3 FRETdye.

FIG. 13 illustrates an individual emission spectrum for a Cy5-X-Cy3 FRETdye.

FIG. 14 illustrates an individual emission spectrum for a Cy5-Y-Cy3 FRETdye.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is generally directed to labeled compounds,compositions containing such labeled compounds, and methods of usethereof, in particular in analytical reactions. In certain aspects, thepresent invention is directed to compounds comprising detectable labelsthat comprise a plurality of chromophores (e.g., fluorophores) thatundergo Förster resonance energy transfer (FRET), and these labeledcompounds are particularly useful in certain analytical reactions, e.g.,for monitoring progress of the reaction, substrate processing, and/orproduct formation. In other aspects, the present invention is directedto compounds comprising detectable labels comprising at least onechromophore that undergoes FRET with at least one other chromophore onat least one other compound. In some aspects, the present invention isdirected to analytical reactions comprising one or more labeledcompounds that undergo intermolecular and/or intramolecular FRET. Theuse of excitation of chromophores and monitoring of their emissions arewell known to those of skill in the art. Briefly, a chromophore in itsexcited state (e.g., due to exposure to excitationradiation/illumination) can emit energy to return to its ground state,and the emission spectra of a given chromophore has a set ofcharacteristics (e.g., wavelength of peaks, intensity of peaks, numberof peaks, shape of peaks, etc.) that can be used to detect thechromophore during an analytical reaction. When different chromophoresare used to label different molecules, the emission spectrum thatcharacterizes a particular chromophore can serve as a proxy for thepresence of the particular molecule labeled therewith. A chromophore inits excited state can also transfer energy by a nonradiative, long-rangedipole-dipole coupling mechanism to a second chromophore in closeproximity, and this transfer is referred to as “FRET.” This transfer isrelated to the overlapping spectra of the two chromophores, where theemission spectrum of the initially excited chromophore overlaps theabsorption spectrum of the second chromophore so that there is energytransfer from the excited chromophore to the second chromophore. If theefficiency of the energy transfer is high (e.g., ˜90-100%), the emissionspectrum produced upon excitation of the two chromophores primarilycomprises detectable emissions from the second chromophore.

Detectable labels that undergo FRET are termed “FRET labels” herein, andtypically comprise at least two chromophores that engage in FRET suchthat at least a portion of the energy absorbed by at least one “donorchromophore,” e.g., during excitation illumination, is transferred to atleast one “acceptor chromophore,” which emits at least a portion of thetransferred energy as a detectable signal contributing to an emissionspectrum. “FRET pair” refers to two chromophores that undergo FRET,e.g., one being the donor and one being the acceptor. A FRET label maycomprise a single FRET pair, or multiple FRET pairs, as describedelsewhere herein. In certain embodiments, a donor dye is excited withthe laser source at its maximum absorbance wavelength, and an acceptordye absorbs the emission from the donor with a given efficiency andemits the energy at a different wavelength, preferably a longerwavelength. It will be understood that the designations “donorchromophore” and “acceptor chromophore” are based on the direction ofenergy transfer between two chromophores, and in some embodiments, inparticular those in which a FRET label comprises three or morechromophores, a single chromophore may serve as both a donor chromophoreand an acceptor chromophore. For example, energy may be transferred froma first chromophore (donor) to a second chromophore (acceptor) that hasa higher emission wavelength, and the second chromophore (now a donor)can pass energy onto a third chromophore (acceptor) with an even higheremission wavelength. FRET labels comprising more than two chromophorescan be beneficial in various ways. For example, their use can increasethe difference in the excitation/absorption wavelength and the observedemission wavelength, which can reduce interference of the excitationradiation with measurement of the emission spectrum. Further, use of aFRET label comprising greater than two chromophores allows greaterflexibility in generating a set of distinctive and unique emissionspectra from different FRET labels comprising the same set ofchromophores, as described below. Although certain preferred embodimentsfocus on FRET labels on a single labeled compound, it is to beunderstood that different chromophores of a FRET label may also resideon different labeled compounds, e.g., resulting in energy transfer whenthe two or more labeled compounds interact in a way that brings thechromophores close enough to one another to allow energy transfer, e.g.,during binding and/or complex formation.

In certain aspects, the chromophores in a FRET label, e.g., in a labeledcompound, are configured to achieve a desired efficiency of the energytransfer (“FRET efficiency”) between the donor and acceptorchromophores, where the desired FRET efficiency is chosen to ensure adesired emission intensity (or range thereof) at one or more emissionwavelengths in the emission spectrum. As used herein, “emissionintensity” refers to the intensity of emitted signal at a givenwavelength, and can generally be related to the height of a peak in anemission spectrum graph, where a relatively higher peak is indicative ofa higher emission intensity and a relatively lower peak is indicative ofa lower emission intensity. FRET efficiency (E) generally refers to theloss in intensity of the donor chromophore emission in the presence ofthe acceptor chromophore, and can be expressed using the followingequation:

${E = {1 - \frac{Q_{da}}{Q_{d}}}},$where Q_(da) is the fluorescence intensity of the donor in the presenceof the acceptor and Q_(d) is the fluorescence intensity of the donor inthe absence of the acceptor. Essentially, the equation provides thefraction of donor fluorescence that is transferred to the acceptorfluorophore.

A desired FRET efficiency, as used herein, is the calculated FRETefficiency based upon the configuration of a FRET label, e.g. within alabeled compound or in a complex comprising multiple labeled compounds,and it will readily be understood that in practice the experimental FRETefficiency may vary somewhat from the desired FRET efficiency. Forexample, in some cases, a FRET label configured to have a desired FRETefficiency experimentally produces an emission spectrum having a smallrange of FRET efficiencies, e.g, within about 15%, or more preferablywithin about 10%, or even more preferably within about 5%, 3%, 2%, or 1%of the desired FRET efficiency. As such, a configuration of chromophoreschosen to achieve a desired FRET efficiency does not necessarily meanthat the FRET efficiency achieved in a given assay will be exactly thedesired efficiency, but that the experimental FRET efficiency may varysomewhat within a range around the desired FRET efficiency. As such, theactual emission intensities achieved with a given molecularconfiguration may also vary somewhat from the desired emissionintensities computed based upon a desired FRET efficiency as the actualemission intensities in an emission spectrum are dependent upon theactual range of FRET efficiencies achieved in a given experiment. Inpractice, such minor variations in FRET efficiencies are not typicallyproblematic. Where multiple labeled compounds are present in a singleanalytical reaction they are designed to have distinguishable emissionspectra even in the presence of minor variations in FRET efficiency. Theemission spectra of multiple labeled compounds can also be compared toone another to confirm the identity of a given labeled compound orassociation between two or more labeled compounds in the reaction. Forexample, in some cases the relative emission intensities at variouswavelengths compared between emission spectra are more informative inidentifying the source of a particular emission spectrum than theabsolute emission intensities that characterize it.

In certain preferred embodiments, the configuration of the chromophores(e.g., spacing between them) in the compound or complex determines theFRET efficiency, and therefore the emission spectrum. For example, insome FRET labels a spacing of about 2 nm allows very high FRETefficiency, while a spacing of about 9 nm results in a relatively lowFRET efficiency. Other factors that influence FRET efficiency includethe spectral overlap of the donor emission spectrum and the acceptorabsorption spectrum, and the relative orientation of the donor emissiondipole moment and the acceptor absorption dipole moment. Thisinformation is available to the ordinary practioner for a large varietyof chromophores, allowing substantial flexibility in choosing FRETlabels for various applications of the instant invention.

In certain embodiments in which the FRET efficiency is less than 100%,at least two chromophores in a FRET label emit detectable signals thatcontribute to the resulting multi-spectral emission spectrum, e.g.,represented by at least two “peaks” characterized by their wavelengthand intensity. In general, as the FRET efficiency increases, theemission intensity at the donor chromophore's emission wavelengthdecreases and the emission intensity at the acceptor chromophore'semission wavelength increases. As such, two FRET labels that eachcomprise the same set of chromophores can have distinct emission spectraif each is configured to ensure a distinct FRET efficiency or rangethereof. For example, if a first FRET label has a higher FRET efficiencythan a second FRET label, the emission spectrum corresponding to thefirst FRET label will have a relatively lower intensity peak at theemission wavelength of the donor chromophore and a relatively higherintensity peak at the emission wavelength of the acceptor chromophorethan does the second FRET label. For example, the intensity of the firstFRET label at the emission wavelength of the donor chromophore may beless than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of that ofthe second FRET label; and the intensity of the second FRET label at theemission wavelength of the acceptor chromophore may be less than 90%,80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of that of the first FRETlabel. The differences between emission intensities at donor or acceptoremission wavelengths for two different FRET labels can also be expressedas ratios of the intensities for each label, e.g., 10:1, 8:1, 6:1, 4:1,3:1, 2:1, 1:2, 1:3, 1:4, 1:6, 1:8, or 1:10 at a given wavelength. Inthis way, FRET labels comprising the same set of chromophores can beconfigured such that each has a distinctive emission spectra based atleast on emission intensities, even if emission wavelengths are thesame. In certain aspects, a single FRET pair may be used to provide atleast about 2-10 different emission spectra based on the orientation ofthe chromophores relative to one another. Further, FRET labels havingmore than two chromophores can provide even more different emissionspectra based on the orientation of the chromophores with respect to oneanother, and therefore the relative FRET efficiencies of each transferevent within the label.

Of particular interest is the structure within the labeled compounds(e.g., linker(s)) that maintains the proper configuration of thechromophores to result in a desired FRET efficiency (and thereforeemission spectrum) with no or only a minimum of undesirable effects tothe analytical reaction under examination. As is well known in the art,the efficiency (E) of the transfer of the excitation energy from thedonor to the acceptor depends on the donor-to-acceptor separationdistance r with an inverse 6^(th) power law due to the dipole-dipolecoupling mechanism: E=1/[(1+(r/R₀)⁶], where r is the distance betweenthe donor and acceptor and R₀ is the Förster distance of the donor andacceptor pair at which the FRET efficiency is 50%. The efficiencyrapidly increases to 100% as the separation distance decreases below R₀,and conversely, decreases to zero when r is greater than R₀. (For areview of FRET microscopy, see, for example,www[dot]olympusfluoview[dot]com/applications/fretintro[dot]html (where“[dot]” indicates a period in the web address), which is incorporatedherein by reference in its entirety for all purposes.) When r isapproximately 50% of R₀, the resonance energy transfer efficiency isnear the maximum. When the donor-acceptor distance exceeds the R₀ valueby 50%, the slope of the curve is so shallow that longer separationdistances are not resolved. Alternatively, when the donor and acceptorradius (r) equals the Förster distance, then the transfer efficiency is50%. At this separation radius, half of the donor excitation energy istransferred to the acceptor via resonance energy transfer, while theother half is dissipated through a combination of all the otheravailable processes, including fluorescence emission. Thus, anappropriate length of r can be used to modulate the energy transferefficiency and thus create a unique emission spectrum.

FRET labels with multi-spectral properties can be used alone, e.g., on asingle labeled compound or on multiple interacting labeled compounds;with other FRET labels having multi-spectral properties; with other FRETlabels not having multi-spectral properties; with non-FRET labels; or acombination thereof, e.g., in a single analytical reaction. Non-FRETlabels used with FRET labels may comprise the same or differentchromophores as the FRET labels, as long as the presence of thechromophore in the non-FRET labels does not interfere with the energytransfer within the FRET labels. Emission spectra from different labelsin a single analytical reaction can be distinguished using variouscriteria including, but not limited to, emission wavelength(s), emissionintensity(s), spectral shape, or a combination thereof. As describedabove, differences in emission intensity at a given wavelength fordifferent FRET labels can also be expressed in terms of percentdifferences or ratios. Detailed descriptions of certain examples ofcriteria for distinguishing emission spectra are provided in U.S. PatentPublication No. 2009/0024331, the disclosure of which is incorporatedherein by reference in its entirety for all purposes. Alternate labelingstrategies that can also be used with the methods and compositionsdescribed herein include those provided in U.S. Patent Publication No.2009/0208957, which is incorporated herein by reference in its entiretyfor all purposes.

In certain aspects, a composition of the invention comprises a pluralityof FRET-labeled compounds in a single analytical reaction. In preferredembodiments, the configuration and/or types of the chromophores in eachcompound results in the production of distinctive emission spectra,wherein each FRET-labeled compound has an emission spectrum that isdistinguishable from the emission spectrum of every other FRET-labeledcompound in the analytical reaction, thereby enabling unambiguousidentification of each FRET-labeled compound. In certain embodiments,the multiple different labeled compounds comprise the same set ofchromophores, but have a different configuration and therefore differentemission spectra based at least in part on different FRET efficienciesresulting in variations in emission intensity at one or more wavelengthsand, in certain preferred embodiments, not due to a change in theemission wavelength(s). In other words, two or more different emissionspectra can comprise the same number of peaks at the same wavelengths,but still be distinct from one another based on the emission intensityat those peaks rather than the emission wavelengths at which the peaksare present. In other embodiments, multiple different labeled compoundscan comprise different sets of chromophores, and their differentemission spectra can be distinguished based at least in part onvariations in emission wavelengths and/or emission intensities. Incertain embodiments, multiple different labeled compounds comprise some,but not all, of the same chromophores. For example, a single reactionmixture can contain a first labeled compound comprising a FRET labelconsisting of two chromophores, and a second labeled compound comprisingthe two chromophores in the first labeled compound and a thirdchromophore that serves as an acceptor for the emission from the firsttwo chromophores, emitting at an additional or different emissionwavelength. In some such embodiments, the emission spectra from thefirst and second labeled compounds may be distinguished not only bychanges in intensity at emission wavelengths corresponding to the firsttwo chromophores, but also by the existence of a third emissionwavelength in the emission spectra of the second labeled compound.Additional, optically distinctive labels can be designed by changing theconformation of the first and second FRET labels to change the FRETefficiency within the two chromophores in the first label or between thethree chromophores in the second label.

In some aspects, the compositions of the invention can further comprisenon-FRET-labeled compounds that have emission spectra that are distinctfrom the emission spectra of the FRET-labeled compounds of theinvention. In some embodiments, a single labeled compound can compriseboth a FRET label and a non-FRET label, where the non-FRET label has anemission spectrum that is independent of the FRET label. In certainembodiments, FRET-labeled compounds may be combined withnon-FRET-labeled compounds (e.g., comprising one or more chromophores)in a single analytical reaction, with identification of the reactiveportion of the labeled compound based at least in part on a comparisonof the emission spectra generated from the label portions. For example,a first labeled compound can be labeled with chromophore A, a secondlabeled compound can be labeled with chromophore B, a third labeledcompound can be labeled with the A-B FRET pair in a first orientationthat results in 30% FRET efficiency, and a fourth labeled compound canbe labeled with the A-B FRET pair in a second orientation that resultsin 70% FRET efficiency. An prophetic graphical representation of thevarious emission spectra that can result from these four differentlabeled compounds is provided in FIG. 1 . The labeled compound labeledwith only chromophore A (“Chromophore A”) produces a single peak of 100%relative emission intensity at a first wavelength and the labeledcompound labeled with only chromophore B (“Chromophore B”) produces apeak of 100% relative emission intensity at a second wavelength. Thelabeled compound labeled with the FRET pair having 30% FRET efficiency(“30% FRET”) produces two peaks, one of relatively high emissionintensity (˜70% relative emission intensity) at the first wavelength andone of relatively low emission intensity (˜35% relative emissionintensity) at the second wavelength. The labeled compound labeled withthe FRET pair having 70% FRET efficiency (“70% FRET”) produces twopeaks, one of relatively low emission intensity (˜30% relative emissionintensity) at the first wavelength and one of relatively high emissionintensity (˜72% relative emission intensity) at the second wavelength.These four prophetic emission spectra are easily distinguishable fromone another, thereby allowing the ordinary practitioner to unambiguouslyidentify the labeled compound, and in particular the reactive portionthereof, by virtue of the characteristics of a detected emissionspectrum. In this way, four different emission spectra are produced in asingle analytical reaction using only two chromophores, two of theemission spectra being produced from FRET pairs having differentconformations and therefore different FRET efficiencies, and two of theemission spectra being produced from non-FRET labels comprising only oneof the chromophores in the FRET pairs.

In certain aspects, the present invention is directed to labeledcompounds useful as analogs to naturally occurring reaction componentsin analytical reactions, including but not limited to, binding assays(e.g., antibody assays), nucleic acid sequencing, protein sequencing,methylation mapping, secondary structure analysis, enzyme assays,kinetic studies, assays that monitor conformation changes ofmacromolecules or macromolecular complexes, and the like. For example,the FRET labels of the invention can be used to differentially labeltRNA molecules during translation in order to determine the sequence ofamino acids incorporated into a nascent polypeptide chain, to detectbinding of a ligand to a receptor, to measure antigen binding to anantibody, to monitor the rate of an enzymatic reaction, and to determinethe sequence of polymers during synthesis. In certain preferredembodiments, at least one reaction component, labeled or unlabeled, isimmobilized or otherwise confined at a reaction site that is monitoredfor the presence of labeled reactants. In certain preferred embodiments,a single reaction site is optically resolvable from other reaction sitesin an analytical reaction such that a single molecule of a labeledcompound can be detected and distinguished from other labeled compoundspresent in the reaction. Such analytical reactions may comprise labeledcomponents having intramolecular FRET labels, intermolecular FRETlabels, non-FRET labels, or combinations thereof. Certain embodiments ofsuch analytical reactions are provided in U.S. provisional applicationNo. 61/186,661, filed Jun. 12, 2009; U.S. provisional application No.61/186,645, filed Jun. 12, 2009; and U.S. Ser. No. 12/635,618, filedDec. 10, 2009, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

In certain embodiments, the present invention is directed to labeledcompounds useful as analogs to naturally occurring nucleosidetriphosphates or previously described analogs in a variety of differentapplications, including particularly, analytical nucleic acid analysessuch as genotyping, sequencing, and other characterization andidentification analyses. For example, in certain embodiments anucleotide analog is a nucleotide tetraphosphate, a nucleotidepentaphosphate, a nucleotide hexaphosphate, a nucleotide septaphosphate,or a nucleotide octophosphate. In preferred embodiments, the labeledcompounds are used in template-directed sequencing reactions and theirincorporation into a nascent nucleic acid strand (e.g., DNA or RNAstrand) is monitored in real-time. In some embodiments, all nucleotideanalogs in an analytical reaction are FRET-labeled compounds. In otherembodiments, a first subset of nucleotide analogs are labeled withFRET-labels and a second subset of nucleotide analogs are labeled withnon-FRET-labels in a single analytical reaction, e.g., atemplate-directed sequencing reaction. In some embodiments, a singlenucleotide analogs is labeled with both a FRET label and a non-FRETlabel. For example, a sequencing reaction may comprise two differentnucleotide analogs labeled with FRET labels having the same FRET pairbut different FRET efficiencies, and the other two nucleotide analogseach labeled with a different one of the chromophores that make up theFRET pair in the FRET-labeled nucleotide analogs. As such, only twochromophores would be required to differentially label four differentnucleotide analogs, two with FRET labels and two with non-FRET labels.Therefore, two chromophores can be used to generate four distinctemission spectra in an analytical reaction. In some embodiments, morethan one FRET-labeled nucleotide analog in a reaction has at least oneor two chromophores in common. In some embodiments, chromophores in aFRET-labeled compound are fluorophores.

The compounds and methods herein may further comprise various aspects ofthe following patent applications and patents: U.S. Ser. No. 61/069,247,filed Mar. 13, 2008; U.S. Ser. No. 12/241,809, filed May 15, 2008; U.S.Pub. No. 2008/0241866 A1; U.S. Pat. No. 7,405,281; U.S. Pat. No.7,056,676; and US Pat. No. 5,688,648, all of which are incorporatedherein by reference in their entireties for all purposes.

I. Compounds

In certain aspects, the present invention is directed to FRET-labeledcompounds comprising at least two chromophores and uses thereof. Incertain aspects, the labeled compound is configured to providesubmaximal FRET efficiency upon excitation illumination, and thissubmaximal FRET efficiency is chosen to promote production of a desiredemission intensity at one or more emission wavelengths that isdetectably different than that produced when FRET efficiency ismaximized, as further described elsewhere herein. For example, thedistance between the chromophores in the FRET label can be chosen toprovide a desired FRET efficiency upon excitation illumination, and thisdistance will produce a distinct and identifiable emission spectra(e.g., a multi-spectral emission spectra where emissions at two or moredifferent wavelengths are detectable) based in part of the emissionintensities at the emission wavelengths of both the donor and acceptorchromophores. As such, the conformation of the labeled compounds, and inparticular the orientation of the chromophores with respect to eachother, is of particular interest. For purposes of description, thelabeled compound comprises a label portion and a reactant portion, thereactant portion denoting the portion of the labeled compound thatserves as the reactant in the reaction of interest, with or without thelabel group. For example, in nucleic acid reactions utilizingfluorescently labeled nucleotide analogs as the labeled compound, thelabel portion that includes the fluorescent dye component(s) isconnected to the reactant portion comprising a nucleotide analog. Asused herein, the term “nucleotide analog” refers generally tonucleosides, nucleotides, and analogs and derivatives thereof. Indescribing certain labeled compounds of the invention as nucleotideanalogs, it is meant that in a particular application, the compounds orcompositions function in a manner similar to or analogous to naturallyoccurring nucleoside triphosphates (or nucleotides), and does nototherwise denote any particular structure to such compounds. Forexample, in certain embodiments a nucleotide analog for use as a labeledcompound of the invention comprises a polyphosphate with at least 2, 3,4, 5, 6, 7, 8, 9, or 10 phosphate groups. Similarly, for a bindingassay, a first binding partner may be immobilized on a surface and asecond binding partner that specifically associates with the firstbinding partner can comprise a label portion that includes thefluorescent dye component(s) connected to a reactant portion comprisinga ligand for the first binding partner. In some such embodiments, thefirst binding partner also comprises a FRET or non-FRET label portion.In certain embodiments, FRET occurs between a label portion on the firstbinding partner and a label portion on a second binding partner. Suchbinding partners include, e.g., antibodies and antigens, receptors andligands, enzymes and substrates, complementary nucleic acid molecules,nucleic acid binding sites and proteins that bind to them (e.g.,histones, transcription factors, etc.), and the like.

Typically, a labeled compound of the invention is configured to ensure adesired FRET efficiency (or range thereof) between at least twochromophores such that, upon excitation illumination, a distinctive andidentifiable emission spectrum is generated based upon emissionintensity at one or more emission wavelengths. In preferred embodiments,multiple compounds labeled with the same set of chromophores (e.g., FRETpair) are configured such that each has a unique orientation of thechromophores with respect to one another, resulting in the production ofdistinctive and identifiable emission spectra for each labeled compound.Typically, the label portion of such labeled compounds provides linkagebetween the chromophores of sufficient lengths and rigidities tomaintain the desired orientation of the chromophores with respect to oneanother (e.g., during an analytical reaction) such that a distinctemission spectrum will be reliably produced upon excitationillumination.

In a first aspect, the maintenance of the desired relative orientationof the chromophores (e.g., distance between them) may be characterizedas a function of the desired reduction in maximal FRET efficiency uponexcitation illumination as compared to similar molecules in which theorientation is selected to optimize FRET efficiency. Put another way, arange of distances between the donor and acceptor chromophores can bechosen to ensure inefficient energy transfer (and therefore submaximalFRET efficiencies), which produce distinct emission spectra thattypically include peaks at wavelengths corresponding to both donor andacceptor emission wavelengths. For example, the chromophores may beoriented to have a FRET efficiency that is about 0%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or95% of the maximal FRET efficiency between a set of chromophores werethey oriented to maximize energy transfer between the chromophores. In asimple, ideal, two-chromophore FRET label, a configuration that ensured0% FRET efficiency would result in no energy transfer between donor andacceptor chromophores, so the emission intensities and wavelengths woulddepend on how well the excitation illumination can excite eachchromophore independently and what the resulting emission would be fromeach. Typically, a peak of high intensity would be produced by emissionfrom the donor chromophore, and no peak (or a very small peak) would beproduced by emission from the acceptor chromophore, e.g., whereexcitation illumination was of a wavelength that excited the donor butnot the acceptor. A configuration that promoted 100% FRET efficiencywould result in complete transmission of all excitation energy from thedonor chromophore to the acceptor chromophore, so the emission spectrawould only contain signal resulting from emission from the acceptorchromophore. A configuration that promoted 50% FRET efficiency wouldresult in the intensity of the donor emission being reduced by half, anda resulting increase in the intensity of the acceptor, resulting in anemission spectrum comprising significant emission signal from both thedonor and acceptor. In this way, the orientation of the chromophores canbe adjusted to achieve a desired FRET efficiency for a given FRET label.

FRET-labels comprising more than two chromophores are capable ofproducing more complex emission spectra, depending on the FRETefficiency between each pair of chromophores between which energytransfer occurs. In some embodiments, the FRET labels are designed toallow some level of emission from all chromophores to contribute to theemission spectrum, and in other embodiments the labels are designed torestrict detectable emission to only a subset of chromophores. The FRETefficiency between each pair of chromophores undergoing energy transfercan be the same or can differ within a single FRET label. As such, in athree-chromophore FRET label, the FRET efficiency between a first andsecond chromophore can be the same or different from the FRET efficiencybetween the second chromophore and a third chromophore. As will be clearto one of ordinary skill in the art, the number of chromophores and FRETefficiency between them can be adjusted to provide a vast number ofdistinct and identifiable emission spectra by virtue of the resultingemission intensities, even when each emission spectrum includes peaks atone or more of the same wavelengths. As such, in some preferredapplications, a plurality of multiple emission intensities at aplurality of emission wavelengths allows the various labeled compoundsin a reaction to be reliably distinguished from one another.

In another aspect, the labeled compounds of the invention arecharacterized by the specific distances provided between thechromophores (e.g., in a FRET pair) in the label portion of a singlelabeled compound or within a complex comprising multiple labeledcompounds. Because of differences in the relative flexibility ofdifferent linkages, such distances are generally stated in terms of anoperating or functional distance, e.g., the average maintained distancebetween the chromophores. In the case of linear linkages, such distancesmay be provided using polymers or other linear structures that havepersistence lengths of the desired distances. Alternatively, somelinkages may provide a spatial separation based upon the volume of thelinkage, e.g., PEG linkers may exist as a random coil and polyprolineforms a type II trans helix, both of which can provide a relativelyconsistent spatial separation between the chromophores.

While precise distances or separation may be varied for differentreaction systems to obtain optimal results, in many cases it will bedesirable to provide a linkage that maintains a distance of about 2-8 nmbetween chromophores in a FRET pair. The specific spacing between thechromophores will vary depending on the chromophores used and thedesired FRET efficiency (0-100%).

A number of linkers may be employed that will provide the desiredconformation of the FRET label chromophores within a labeled compound orcomplex of multiple labeled compounds, e.g., including the separationbetween chromophores in a FRET pair, the distance between a chromophoreof a FRET pair and a reactant portion, or the distance between achromophore in a first labeled compound and a chromophore in a secondlabeled compound when the first and second compounds are bound to orotherwise associated with one another. In general, a linker comprisingone or more chromophores can be linear or branched, and multiple linkersmay be utilized in a single labeled compound. For example, a singlelinker may be bound to the terminal phosphate of a nucleotide analog,and this linker may branch into two arms, wherein each arm comprises asingle chromophore of a FRET pair, and wherein the orientation of thetwo arms ensures a given distance between the two chromophores, therebyensuring a desired FRET efficiency upon excitation illumination. Inother embodiments, a linear linker may contain both chromophores, withthe portion of the linker between them designed to ensure a givenorientation between them, and therefore a desired FRET efficiency. Inyet further embodiments, multiple linear and/or branched linkers may bebound to different portions of a polyphosphate chain extending from thenucleotide analog, and each of these may comprise one or morechromophores in a desired orientation.

A wide variety of linkers and linker chemistries are known in the art ofsynthetic chemistry and may be employed in constructing the labelportions and coupling them to the reactant portions of the compounds ofthe invention. For example, such linkers may include organic linkerssuch as alkane or alkene linkers of from about C2 to about C20, orlonger, polyethyleneglycol (PEG) linkers, aryl, heterocyclic, saturatedor unsaturated aliphatic structures comprised of single or connectedrings, amino acid linkers, peptide linkers, nucleic acid linkers, PNA,LNAs, or the like or phosphate or phosphonate group containing linkers.In preferred aspects, alkyl, e.g., alkane, alkene, alkyne alkoxy oralkenyl, or ethylene glycol linkers are used. Some examples of linkersare described in Published U.S. Patent Application No. 2004/0241716;U.S. Ser. No. 61/069,247, filed Mar. 13, 2008; and U.S. PatentPublication No. 2009/0233302, all of which are incorporated herein byreference in their entireties for all purposes. Additionally, suchlinkers may be selectively cleavable linkers, e.g., photo- or chemicallycleavable linkers or the like, many types of which are known by those ofordinary skill in the art.

In certain embodiments alkyl linkers may be used to provide a usefuldistance between the chromophores. For example, longer amino-alkyllinkers, e.g., amino-hexyl linkers, are generally sufficiently rigid tomaintain a desired spacing. In certain aspects providing linkers withdesired functional lengths can involve the use of more rigid chemicalstructures in such linkers. Typically, such rigid structures includelaterally rigid chemical groups, e.g., ring structures such as aromaticcompounds, multiple chemical bonds between adjacent groups, e.g., doubleor triple bonds, in order to prevent rotation of groups relative to eachother, and the consequent flexibility that imparts to the overalllinker. Alternatively or additionally, secondary chemical structures maybe used to impart rigidity, including, for example helical structures,sheet structures, and the like, as well as structures that employcooperative molecules in providing rigidity, e.g., complementarymolecular structures. Other examples of the linkers of the inventioninclude oligopeptide linkers, and in particular, oligoproline orpolyproline linkers that include ring structures. (See, e.g, Schuler, B.(2005) Proc. Natl. Acad. Sci. 102(8):2754-2759, incorporated byreference herein in its entirety for all purposes.)

As noted, some linkers according to the invention derive rigiditythrough the internal chemical structure of the linker molecules. Forexample, linker molecules may derive their rigidity through a reductionin the number of single bonds that can yield points of rotation, andthus, flexibility in the linker. As such, the linkers will typicallycomprise double bonds, triple bonds or ring structures, which willprovide the increased rigidity. Examples of double and/or triple bondedlinker structures include, for example, conjugated alkynes, conjugatedalkenes, aryl alkynes, and the like. While illustrated as polymericstructures of repeating monomeric subunits, it will be appreciated thatthe linkers of the invention may comprise mixed polymers of differingmonomeric subunits.

The linkers used in the context of the invention may additionally oralternatively derive rigidity from secondary, tertiary, or evenquaternary structures. For example, in some cases, polypeptide linkersmay be employed that have helical or other rigid structures. Suchpolypeptides may be comprised of rigid monomers, e.g., as in theoligoproline or polyproline linkers noted above, which derive rigidityboth from their primary structure, as well as from their helicalsecondary structures, or may be comprised of other amino acids or aminoacid combinations or sequences that impart rigid secondary or tertiarystructures, such as helices, fibrils, sheets, or the like. By way ofexample, polypeptide fragments of structured rigid proteins, such asfibrin, collagen, tubulin, and the like may be employed as rigid linkermolecules.

In a related aspect, double-stranded nucleic acids can be used toprovide both the requisite length and rigidity as a linker. Similarly,related structures, such as double-stranded peptide nucleic acids (PNAs)or DNA/PNA hybrid molecules may be employed as the linkers. By way ofillustration, the persistence length of double-stranded nucleic acids,i.e., the length up to which the structure behaves more rod-like thanrope-like, is approximately 50 nm, allowing for facile construction ofrigid linkers up to and even beyond this length. In certain preferredaspects, a nucleic acid linker that comprises a double-stranded portionto impart rigidity is used as a linker group, e.g., between thechromophores or between one or more chromophores and the reactantportion of the labeled compound. Such double-stranded nucleic acids maycomprise distinct but complementary nucleic acid strands that arehybridized together, where one or both strands bear a chromophore.Alternatively, the nucleic acid linker may comprise a single moleculewith complementary portions, such that the molecule self hybridizes toform a hairpin loop structure, where the label component is provided ata point on the loop, distal to the reactant portion. The use of nucleicacid linker structures provides advantages of ease of synthesis of thelabeled linker, using conventional DNA synthesis and dye couplingtechniques, and resultant control of linker length, e.g., approximately0.3 nm of distance imparted for each added monomer in the linkerportion. Consequently, one can easily adjust the length of the linker toaccommodate various spacing between the chromophores and the reactantportion, or between the chromophores themselves. Additionally, theability to adjust the rigidity of the linker, in real time providesinteresting reaction control elements, e.g., by adjusting the integrityof the hairpin structure by modifying the hybridization conditions forthe linker, e.g., adjusting salt, temperature, or the like. Theselinkers are also readily coupled to nucleotide analogs, whether coupledthrough groups on the nucleobase, the ribosyl moiety, or through one ofthe phosphate groups (e.g., alpha, beta, gamma, or others in the case oftetra, penta, or hexa phosphate analogs, or others). Further descriptionof such nucleic acid linkers is provided, e.g., in U.S. Ser. No.61/069,247, filed Mar. 13, 2008 and U.S. Patent Publication No.2009/0233302, both of which are incorporated herein by reference intheir entireties for all purposes.

In certain aspects, the label portion of a labeled compound comprises atleast one detectable labeling moiety, or more than one detectablelabeling moiety, and in certain preferred embodiments, and as describedabove, the label portion of a labeled compound comprises at least twodetectable labeling moieties that preferably can form a FRET paircoupled to the reactant portion via at least one linking group.Detectable labeling moieties generally denote chemical moieties thatprovide a basis for detection and identification of the reactant portionof the labeled compound. In preferred aspects, the detectable labelingmoieties comprise optically detectable moieties, including luminescent,chemiluminescent, fluorescent, fluorogenic, chromophoric and/orchromogenic moieties (with fluorescent and/or fluorogenic labels beingparticularly preferred), but such labels may also impart additionalproperties to the labeled compounds of the invention, e.g., a detectableelectrical or electrochemical property, or a different physical orspatial property. A multitude of labeling moieties is known to those ofordinary skill in the art, some of which are described herein.

Chromophores useful with the compounds, compositions, and methods of theinvention may be fluorescent dyes, non-fluorescent dyes, or the like.Examples of suitable chromophores include, but are not limited to,fluorescein and its derivatives, rhodamine-based dyes, and thecyanine-based dyes such as isothiocyanines, merocyanines,indocarbocyanines (e.g., Cy3 and Cy3.5), indodicarbocyanines (e.g., Cy5and Cy5.5), indotricarbocyanines (e.g., Cy7), thiazole orange, oxazoleyellow, the Alexa® dyes (e.g., Alexa 488, 555, 568, 647, and 660), CYA(3-(epsilon-carboxy-pentyl)-3′ethyl-5,5′dimethyloxacarbocyanine), andthe like (see Mujumdar, et al Bioconjugate Chem. 4(2):105-111, 1993;Ernst, et al, Cytometry 10:3-10, 1989; Mujumdar, et al, Cytometry10:1119, 1989; Southwick, et al, Cytometry 11:418-430, 1990; Hung, etal, Anal. Biochem. 243(1):15-27, 1996; Nucleic Acids Res.20(11):2803-2812, 1992; Mujumdar, et al, Bioconjugate Chem. 7:356-362,1996; Southwick and Waggoner, U.S. Pat. No. 4,981,977, issued Jan. 1,1991, all of which are incorporated herein by reference in theirentireties for all purposes). These and other such chromophores arereadily commercially available, e.g., from the Amersham Biosciencesdivision of GE Healthcare, and Molecular Probes/Invitrogen/LifeTechnologies Inc. (Carlsbad, Calif.)., and are described in ‘TheHandbook—A Guide to Fluorescent Probes and Labeling Technologies, TenthEdition’ (2005) (available from Invitrogen, Inc./Molecular Probes/LifeTechnologies, and incorporated herein by reference in its entirety forall purposes). A variety of other fluorescent and fluorogenic labels foruse with nucleoside polyphosphates, and which would be applicable to thecompounds of the present invention are described in, e.g., PublishedU.S. Patent Application No. 2003/0124576, the full disclosure of whichis incorporated herein in its entirety for all purposes. In certainpreferred embodiments, the chromophores in a FRET pair are fluorophores.

Any of a number of fluorophore combinations can be selected for use inthe present invention (see for example, Pesce et al,. eds, FluorescenceSpectroscopy, Marcel Dekker, New York, 1971; White et al., FluorescenceAnalysis: A practical Approach, Marcel Dekker, New York, 1970; Handbookof Fluorescent Probes and Research Chemicals, 6th Ed, Molecular Probes,Inc., Eugene, Oreg., 1996; which are incorporated herein by referencereference in their entireties for all purposes). In general, a preferreddonor fluorophore is selected that has a substantial spectrum of theacceptor fluorophore. Furthermore, it may also be desirable in certainapplications that the donor have an excitation maximum near a laserfrequency such as Helium-Cadmium 442 nM, Argon 488 nM, Nd:YAG 532 nm,He—Ne 633 nm, etc. In such applications the use of intense laser lightcan serve as an effective means to excite the donor fluorophore. Incertain preferred embodiments, the acceptor fluorophore has asubstantial overlap of its excitation spectrum with the emissionspectrum of the donor fluorophore. In some cases, the wavelength maximumof the emission spectrum of the acceptor moiety is preferably at least10 nm greater than the wavelength maximum of the excitation spectrum ofthe donor moiety. Additional examples of useful FRET labels include,e.g., those described in U.S. Pat. Nos. 5,654,419, 5,688,648, 5,853,992,5,863,727, 5,945,526, 6,008,373, 6,150,107, 6,177,249, 6,335,440, 6,348,596, 6,479,303, 6,545,164, 6,849,745, 6,696,255, and 6,908,769 andPublished U.S. Patent Application Nos. 2002/0168641, 2003/0143594, and2004/0076979, the disclosures of which are incorporated herein byreference for all purposes.

As noted previously, the linkage between the chromophores in a FRETlabel is configured to provide sufficient linker length and structure soas to maintain a sufficient distance between the chromophores, e.g.during detection, thereby ensuring a predictable and distinctiveemission spectrum upon excitation illumination. As noted elsewhereherein, many different kinds of analytical reactions can benefit fromthe use of the FRET labels of the present invention, and the mostbenefit is typically found in those analytical reactions in whichmultiple reactants are to be differentially labeled. For example, in thecontext of nucleic acid (e.g., DNA or RNA) sequencing that employsreal-time detection of the interaction of labeled nucleotides withpolymerase enzymes (e.g., DNA polymerases, RNA polymerases, reversetranscriptases, etc.), one object of the instant invention is to use thesame FRET label to unambiguously identify multiple different labeledcompounds in the reaction based on differences in emission spectrarelated to differences in orientation (e.g., spacing) of thechromophores in the labeled compounds. This is a benefit to the ordinarypractitioner because it allows the use of fewer chromophores to detectthe same number of compounds, which provides flexibility in designingthe analytical reaction. For example, a chromophore that has been shownto negatively impact the quality of an analytical reaction (e.g., bylowering duration, processivity, or fidelity; by damaging reactioncomponents; by having a short half-life in the analytical reaction;etc.) may be omitted in favor of using a FRET label comprisingchromophores with fewer undesirable properties. Likewise, fewerchromophores can be used to label multiple tRNAs during proteintranslation, or to label multiple ligands to a given receptor in abinding assay. Other applications of the compositions and methodsprovided herein will be clear to those of ordinary skill in the artbased upon the teachings provided herein.

The use of FRET labels allows for a high degree of flexibility inchoosing the excitation and emission spectra for the labeled compoundsof the invention, and provides particular advantages for differentiallylabeling various components of an analytical reaction. For example, incertain embodiments across a variety of different compounds, one canutilize a single type of donor chromophore that has a single excitationwavelength, but couple it with multiple different acceptor chromophores(e.g., having an excitation wavelength that at least partially overlapswith the emission spectrum of the donor), where each different acceptorchromophore has an identifiably different emission spectrum. The donorchromophore may be on the same or a different reactant as the acceptorchromophore. For example, in some embodiments the donor chromophore isimmobilized at a reaction site or is attached to a reactant thatinteracts with multiple other reaction components, each of which cancarry a detectably different acceptor chromophore. Alternatively,different donor chromophores whose emission spectra overlap may becoupled with different acceptor chromophores. In alternativeembodiments, the donor and acceptor chromophores are the same formultiple labeled compounds, but the conformation of the labeled compoundvaries, resulting in a different FRET efficiency for each pair ofchromophores in each labeled compound. The emission spectra from eachFRET label can thereby be distinctive from every other, e.g., based onemission intensity at a plurality of emission wavelengths, as describedabove. For example, consider two labeled compounds, both with the sameFRET pair comprising a donor chromophore that emits at a firstwavelength and an acceptor chromophore that emits at a secondwavelength, where the conformation of the first labeled compound resultsin a FRET efficiency of 25% and the configuration of the second labeledcompound results in a FRET efficiency of 75%. Under excitationillumination the FRET pair in the first labeled compound would producean emission spectrum with a large peak (high emission intensity) at thefirst wavelength and a small peak (low emission intensity) at the secondwavelength, while the FRET pair in the second labeled compound wouldproduce an emission spectrum with a small peak at the first wavelengthand a large peak at the second wavelength. As such, even though bothemission spectra comprise peaks at both the first and secondwavelengths, these two emission spectra are distinguishable from oneanother, thereby allowing identification of the reactant portion of thelabeled compound. Likewise, the same two chromophores can be used inadditional labeled compounds having different FRET efficiencies thatresult in spectra that are distinguishable from those of the first andsecond labeled compounds, for example a FRET efficiency that results incomparable peaks at the two wavelengths. In other embodiments, a donorlabel can be present on a first reactant and an acceptor label can bepresent on a second reactant, where binding of the first reactant andthe second reactant bring the labels into such proximity as to permitFRET at a first efficiency, e.g., resulting detectable emissions fromboth the donor and acceptor label. Further, a third reactant comprisingthe acceptor label and capable of binding to the first reactant can alsobe present, where the conformation of the acceptor label on the thirdreactant is different than the conformation of the acceptor label on thesecond reactant. As such, binding of the third reactant to the firstreactant permits FRET at a second efficiency that is different than thefirst, and the differing conformations of the second and third reactantsand resulting different FRET efficiencies upon binding the firstreactant allows identification of the reactant bound based upon theresulting emission spectrum. In yet further embodiments, a donorchromophore may be proximal to but not linked to a first reactant at areaction site and an acceptor chromophore may be attached to a secondreactant that interacts with the first reactant in a manner that bringsthe donor and acceptor chromophores into close proximity to allow FRETto occur between them at a desired efficiency.

The configuration of a variety of different labeled compounds having thesame or similar excitation spectra and multiple different emissionspectra has broad utility in a variety of multiplexed analyses,including for example, multi-color nucleic acid sequencing applications,binding assays, enzymatic assays, and protein sequencing applications.In particular, the use of fewer excitation light sources (e.g., a singleexcitation light source) dramatically reduces engineering constraintsfor excitation/detection systems, and also provides a more uniformanalog structure to potentially provide more predictability and/oruniformity for any biochemistry steps involved in the processes, i.e.,except for differences in the base and the acceptor fluorophore.

Labeled Nucleotide Analogs

In certain aspects, the labeled compounds of the invention provide anucleotide analog comprising a nitrogenous base, a sugar, and apolyphosphate chain, containing phosphorus atoms that are optionallysubstituted at various side positions, and optionally linked at one ormore positions by other than an oxygen atom. Certain embodiments ofsubstituted polyphosphate chains are provided, e.g., in U.S. Pat. No.7,405,281, which is incorporated by reference herein in its entirety forall purposes. Coupled directly or indirectly to the polyphosphate chainis at least one label that can undergo FRET with at least one otherlabel. In certain preferred embodiments, at least two chromophores thattogether form a FRET label are coupled to the polyphosphate chain, theconformation of the nucleotide analog (in particular, the distancebetween the two chromophores in the FRET pair) determinative of the FRETefficiency when exposed to excitation radiation. In certain embodiments,an analytical reaction comprises at least two of such labeled nucleotideanalogs, each comprising a different base and each displaying adifferent FRET efficiency such that the emission spectra upon excitationdistinctly identifies the FRET-labeled nucleotide analog, and thereforethe base therein. For example, although the same FRET pair may label twodifferent nucleotide analogs, the positioning of the chromophores withinthe analog can be adjusted to produce a distinguishable emissionspectrum for each analog, based at least in part on FRET efficiency. Inother embodiments, an analytical reaction comprises at least two of suchlabeled nucleotide analogs, each comprising a different base and thesame type of label that can undergo FRET with another labeled reactantin an analytical reaction, e.g., an enzyme or other protein. Forexample, although the same label is found on both labeled nucleotideanalogs, the conformation (e.g. position, linker structure, and thelike) of the label on each analog is different, resulting in a differentFRET efficiency upon interaction with the other labeled reactant, andtherefore detectably different emission spectra.

In some aspects, the labeled compounds of the invention are analogous tonucleotides. In preferred aspects, the labeled compounds are readilyrecognized and processed by nucleic acid processing enzymes, such aspolymerases. In certain embodiments, the labeled compounds areincorporated into growing polynucleotide strands by polymerase enzymes.In certain aspects, such labeled compounds have incorporationefficiencies that are better than or at least comparable totriphosphate, tetraphosphate, pentaphosphate, or hexaphosphate analogs.

In particular, the labeled nucleotide analogs of the invention areparticularly useful as substrates for polymerase enzymes inpolynucleotide synthesis and particularly, template-dependentpolynucleotide synthesis, e.g., DNA polymerases, i.e., Taq polymerases,E. coli DNA Polymerase I, Klenow fragment, reverse transcriptases, Φ29related polymerases including wild type Φ29 polymerase and derivativesof such polymerases, T7 DNA Polymerase, T5 DNA Polymerase, RNApolymerases, and the like, where such synthesis is a component of aprocess for the identification of sequence elements in thepolynucleotide, e.g., individual bases, contiguous sequences ofnucleotides, and/or overall nucleic acid composition, and the like.Another particular advantage of the labeled nucleotide analogs of theinvention is that during incorporation into a synthesized nucleic acidstrand, the chromophores are cleaved from the nucleotide analog by theaction of the polymerase, and thus are not incorporated into thesynthesized strand, resulting in the generation of a natural or “native”strand complementary to the template strand. The removal of thechromophores provides a number of benefits including, for example, theavoidance of any steric interference on a subsequent incorporationevent. Such steric interference can result from, e.g., bulky orchemically incompatible label groups that can interfere with the actionof the synthesis machinery, and can thereby effectively terminate orreduce the rate of continued synthesis. This feature of the methodsdescribed herein is beneficial to other kinds of analytical reactions,as well.

It certain specific embodiments, the invention provides a compositioncomprising a compound of the formula:

wherein B is a natural or non-natural nucleobase or nucleobase analog; Sis selected from a sugar moiety, an acyclic moiety or a carbocyclicmoiety; L is a detectable label optionally including a linker; and R₁ isselected from O and S. R₂, R₃ and R₄ are independently selected from O,methylene, substituted methylene, ethylene, substituted ethylene, wherethe substitutents may include H, F, Cl, OH, NH₂, alkyl, alkenyl,alkynyl, aryl, and heterocycle. In structural terms, the carbons of thesubstituted methylene or ethylene groups will generally comprise thestructure CR′R″, where R′ and R″ are independently selected from H, F,Cl, OH, NH₂, alkyl, alkenyl, alkynyl, aryl, and heterocycle. Examples ofsuch groups include, e.g., CH₂, CF₂, CCl₂, C(OH)(CH₃),C(NH₂)[(CH₂)₆CH₃]) and CH₂CH₂. R₂, R₃ and R₄ are also selected from NH,S, CH(NHR) (where R is H, alkyl, alkenyl, alkynyl, aryl, orheterocycle), C(OH)[(CH₂)_(n)NH2] (n is 2 or 3), C(OH)CH₂R where R is4-pyridine or 1-imidazole. and CNH₂. In preferred aspects, R₂, R₃ and insome cases R₄, are independently selected from O, NH, S, methylene,substituted methylene, CNH₂, CH₂CH₂, C(OH)CH₂R where R is 4-pyridine or1-imidazole.

In addition to the foregoing, R₄ is additionally selected from

R₅, R₆, R₇, R₈, R₁₁, R₁₃, R₁₅ and R₁₇ are, when present, eachindependently selected from OH, BH₃, and S; and R₉, R₁₀, R₁₂, R₁₄ andR₁₆ are independently selected from the same groups as R₂ and R₃, e.g.,O, NH, S, methylene, substituted methylene, CNH₂, CH₂CH₂, C(OH)CH₂Rwhere R is 4-pyridine or 1-imidazole.

The base moiety (“B”) of a labeled nucleotide analogs of the inventionis generally selected from any of the natural or non-natural nucleobasesor nucleobase analogs, including, e.g., purine or pyrimidine bases thatare routinely found in nucleic acids and nucleic acid analogs, includingadenine, thymine, guanine, cytidine, uracil, and in some cases, inosine.For purposes of the present description, reference to nucleotide analogsis based upon their relative analogy to naturally occurring nucleotideanalogs. As such, an analog that operates, functionally, like adenosinetriphosphate, may be generally referred to herein by the shorthandletter A. Likewise, the standard abbreviations of T, G, C, U and I, maybe used in referring to analogs of naturally occurring nucleosides andnucleotides typically abbreviated in the same fashion. In some cases, abase may function in a more universal fashion, e.g., functioning likeany of the purine bases in being able to hybridize with any pyrimidinebase, or vice versa. The base moieties used in the present invention mayinclude the conventional bases described herein or they may include suchbases substituted at one or more side groups, or other fluorescent basesor base analogs, such as 1-N6-ethenoadenosine or pyrrolo-C, in which anadditional ring structure renders the base moiety neither a purine nor apyrimidine. For example, in certain cases, it may be desirable tosubstitute one or more side groups of the base moiety with a labelinggroup or a component of a labeling group, such as one of a donor oracceptor chromophore, or other labeling group. Examples of labelednucleobases and processes for labeling such groups are described in,e.g., U.S. Pat. Nos. 5,328,824 and 5,476,928, each of which isincorporated herein by reference in its entirety for all purposes.

In a labeled nucleotide analog of the invention, the sugar moiety (“S”),in its most preferred aspect, is selected from a D-ribosyl, 2′ or 3′D-deoxyribosyl, 2′,3′-D-dideoxyribosyl, 2′,3′-D-didehydrodideoxyribosyl,2′ or 3′ alkoxyribosyl, 2′ or 3′ aminoribosyl, 2′ or 3′ mercaptoribosyl,2′ or 3′ alkothioribosyl, acyclic, carbocyclic or other modified sugarmoieties. A variety of carbocyclic or acyclic moieties may besubstituted for a sugar moiety, including, e.g., those described inpublished U.S. Patent Application No. 2003/0124576, previouslyincorporated herein by reference in its entirety for all purposes.

For most cases, the polyphosphate chain in the compounds of the presentinvention, e.g., a triphosphate in conventional NTPs, is preferablycoupled to the 5′ hydroxyl group, as in natural nucleosidetriphosphates. However, in some cases, it may be desirable that thepolyphosphate chain is linked to the sugar moiety by the 3′ hydroxylgroup. In certain embodiments, the polyphosphate chain comprises atleast about 3, 4, 5, 6, 7, or 8 phosphates. In certain preferredembodiments, the polyphosphate chain comprises about 4-7 phosphates,with specific embodiments comprising 6 phosphates. Further, while thecompounds of the invention are generally described in terms of includingfour or more phosphorus groups in the phosphorus containing chain, itwill be appreciated that in some instances a three phosphorus atomcontaining chain may be desired.

In certain aspects, the elongated polyphosphate chain, e.g., containingfour or more phosphorus atoms in a linear configuration, is believed toprovide an advantage in the presently described compounds by placinglabeling molecules that may be foreign to nucleotide processing enzymes,e.g., DNA polymerases, away from the relevant portion of the analogand/or away from the active site of the enzyme. Such spacing is believedto reduce the potential for photo-induced damage, e.g., as describedfurther in U.S. Ser. No. 61/116,048, filed Nov. 19, 2008; US Pat. Pub.No. 20070128133, filed Dec. 12, 2005; US Pat. Pub. No. 20070161017,filed Dec. 1, 2006; and US Pat. Pub. No. 20080176241, filed Oct. 31,2007. In addition to providing such distance through the polyphosphatechain, additional linker molecules may be used to provide additionaldistance between the nucleoside portion of the analog and the labelgroups. In particular, while one of the label groups may be directlycoupled to the terminal phosphorus atom of the analog structure, inalternative aspects, it may additionally include a linker molecule toprovide an indirect coupling to the terminal phosphorus atom through,e.g., an alkylphosphonate linkage.

In addition to substitution at the inter-phosphorus linkages, thecompounds of the invention are also optionally substituted at one ormore of the side groups of the phosphorus atoms (or alpha phosphate).Typically, substitution at these side groups, and particularly thosemore distal than the alpha phosphate, will have little negative impacton the incorporation of the analog into a growing nucleic acid strand bya nucleic acid polymerase. In some cases, incorporation of certaingroups at such side groups is expected to provide improved efficiency ofincorporation or processivity of the polymerase enzymes. In particular,boronation of one or more of the subject side groups is expected toprovide such enhanced incorporation. In particularly preferred aspects,the at least one of the oxygen groups on the α phosphate are substitutedwith Boron, and more preferably, the boronated-α-phosphate is the Rpstereo isomer (See, Ramsey-Shaw, et al., Reading, Writing and ModulatingGenetic Information with Boranophosphate Mimics of Nucleotides, DNA, andRNA, (2003) Ann. N.Y. Acad. Sci. 1002:12-29, which is incorporatedherein by reference in its entirety for all purposes). Such α-P-Boranesubstitutions have been shown to improve substrate characteristics fornucleotide analogs, i.e., AZT triphosphate, d4T triphosphate, and 3TCTPin reactions with HIV-1 RT (See, Phillippe Meyer et al., EMBO J. (2000)19:3520-3529, and Jerome Deval, et al., J. Biol. Chem. (2005)280:3838-3846). Additionally, borane modified nucleic acids have beenshown to be resistant to exonucleoase activity (See Ramsey-Shaw et al.supra.). In accordance with certain preferred uses of the compounds ofthe invention, increased stability of a nascent nucleic acid strand toexonuclease activity can be of substantial value, in preventingauto-corrections for misincorporation of a nucleotide during thesynthesis process. Such corrections can yield substantial data analysisproblems in processes that utilize real time observation ofincorporation events as a method of identifying sequence information.

In certain aspects of the invention, the label portion (“L”) of acompound comprises at least one detectable moiety, and in certainpreferred embodiments at least two detectable labeling moieties, and atleast one or more linking groups. In certain preferred embodiments, atleast one of the detectable labeling moieties is indirectly coupled to aphosphorus atom in a polyphosphate chain (e.g., the terminal phosphorusatom) via at least one linking group. In some embodiments, at least oneof the detectable labeling moieties is directly coupled to a phosphorusatom in a polyphosphate chain. In some embodiments, at least twodetectable labeling moieties are indirectly coupled to one or morephosphorus atoms in a polyphosphate chain via at least one linkinggroup. In certain preferred embodiments, the detectable labelingmoieties in a compound of the invention form at least one Försterresonant energy transfer (“FRET”) pair. For example, the FRET pair maybe formed within a single labeled compound (intramolecular FRET) or maybe formed between multiple labeled compounds (intermolecular FRET). Asused herein, intermolecular FRET includes not only FRET between labelson reactants, but also FRET between a labeled reactant and a label on anon-reactant entity or surface in a reaction, e.g., at a reaction site,on a linker, or on a bead, solid surface, or other substrate upon whichthe reaction is localized.

Detectable labeling moieties, and in particular those that can form aFRET pair, are described at length herein. As noted above, the labelingmoieties can comprise optically detectable moieties, includingluminescent, chemiluminescent, fluorescent, fluorogenic, chromophoricand/or chromogenic moieties, with fluorescent and/or fluorogenic labelsbeing particularly preferred. For example, a variety of different labelmoieties are readily employed in nucleotide analogs, and particularly,the compound of the invention. Such groups include fluorescein labels,rhodamine labels, cyanine labels (i.e., Cy3, Cy5, and the like,generally available from the Amersham Biosciences division of GEHealthcare), the Alexa family of fluorescent dyes and other fluorescentand fluorogenic dyes available from Molecular Probes/Invitrogen/LifeTechnologies, Inc., and described in ‘The Handbook—A Guide toFluorescent Probes and Labeling Technologies, Tenth Edition’ (2005)(available from Invitrogen, Inc./Molecular Probes/Life Technologies),semiconductor nanocrystals and other nanoparticle labels (e.g., Qdot®nanocrystals available from Invitrogen, Inc. (Life Technologies)). Avariety of other labeling moieties for use with labeled compounds (e.g.,nucleoside polyphosphates and other biomolecules and reactioncomponents), and which would be applicable to the compounds of thepresent invention are described in, e.g., Published U.S. PatentApplication No. 2003/0124576, the full disclosure of which isincorporated herein in its entirety for all purposes. Additionalexamples of useful FRET labels include, e.g., those described in U.S.Pat. Nos. 5,654,419, 5,688,648, 5,853,992, 5,863,727, 5,945,526,6,008,373, 6,150,107, 6,335,440, 6,348, 596, 6,479,303, 6,545,164,6,849,745 and 6,696,255, and Published U.S. Patent Application No.2003/0143594, the disclosures of which are incorporated herein byreference for all purposes. In certain preferred embodiments, thechromophores in a FRET pair are fluorophores.

For a number of applications, it may be desirable to utilize a differenttype of label portion for each different labeled compound, e.g., eachnucleotide analog that includes a different base, e.g., A, T, G, C, U,I, or analogs or derivatives thereof. In such cases, the label portionsmay be selected so that each label portion has an emission spectrum thatis distinguishable from the emission spectrum of other label portions.Such distinguishable labeled nucleotide analogs provide an ability tomonitor the presence of different labeled compounds simultaneously inthe same reaction mixture. In applications in which multiple differentlabel portions are used to label different compounds, label portions maybe selected to include overlapping excitation spectra, so as to avoidthe necessity for multiple different excitation sources, while providingclearly distinguishable emission spectra.

In certain preferred embodiments, multicomponent label portions areemployed to differentially label at least one or more different reactioncomponents, e.g., different nucleotide analogs. For example, as notedabove, FRET labels may be used in the compounds of the invention. FRETlabels are discussed at length above. In preferred embodiments, themolecular structure of a FRET-labeled compound may comprise one or morelinkers that function to distance the chromophores from the reactiveportion and/or to specifically position the chromophores with respect toone another, e.g., for controlling intramolecular or intermolecular FRETefficiency under excitation illumination.

In some embodiments of the labeled compounds provided herein, a singlelinker comprises a single chromophore in a FRET label, in otherembodiments a single linker comprises multiple chromophores in a FRETlabel, and in yet further embodiments, multiple linkers, each comprisingone or more chromophores in a FRET label, are attached to a singlelabeled component. For example, in a labeled nucleotide analog, one ormultiple linkers may be attached to the polyphosphate chain, eachcomprising at least one chromophore. Such multiple linkers may be boundto the same or different phosphates in the polyphosphate chain. In someembodiments, the configuration of a labeled nucleotide analog isdependent upon which phosphate is linked to the chromophore. Forexample, in certain embodiments a plurality of nucleotide analogs aredistinguished from one another based upon the position of an acceptorchromophore relative to the polyphosphate chain. By positioning theacceptor chromophore at different locations on the polyphosphate chain,the distance to the donor chromophore, and therefore FRET efficiency,can be adjusted. As noted elsewhere herein, the donor and acceptor maybe carried on the same analog, or the donor may be linked to thereaction site or another component with which the nucleotide analog isexpected to interact, e.g., an enzyme such as a polymerase, nuclease,phosphatase, and the like. Linkers of the invention that comprisephosphorus atoms may differ from polyphosphates by virtue of theinclusion of one or more phosphonate groups, effectively substituting anon-ester linkage in the phosphorous containing chain of the nucleotideanalog, with a more stable linkage. Examples of linking groups include,e.g., CH₂, methylene derivatives (e.g., substituted independently at oneor more hydrogens with F, Cl, OH, NH₂, alkyl, alkenyl, alkynyl, etc.),CCl₂, CF₂, NH, S, CH₂CH₂, C(OH)(CH₃), C(NH₂)[(CH₂)₆CH₃], CH(NHR) (R is Hor alkyl, alkenyl, alkynyl, aryl, C(OH)[(CH₂)_(n)NH₂] (n is 2 or 3), andCNH₂. In particularly preferred aspects, methylene, amide or theirderivatives are used as the linkages. Linking groups may be linear,tridentate, or polydentate, and certain preferred but nonlimitingexamples of both linear and tridentate linking groups are provided inFIG. 2 .

In preferred aspects, the compounds include one, two or three suchlinkages, but retain an alpha phosphate that is coupled to the sugar (orcarbocyclic or acyclic) moiety of the nucleotide analog. Retention ofthe alpha phosphate group yields several benefits in the compounds ofthe invention. In particularly preferred embodiments, it permitscleavage of the beta and more distal phosphorus groups and theassociated label from the nucleotide analog by a polymerase enzymeduring processing by that enzyme. Additionally, once processed, thenucleotide analog is more closely analogous (and in some embodiments,identical) to a naturally occurring, processed nucleotide, allowing basedependent hybridization and further minimizing any steric or otherenzyme related effects of incorporation of a highly heterologouscompound into a growing nucleic acid strand.

Examples of certain preferred compounds of the invention include thoseshown below:

The base moiety (“B”) is as described above. The sugar moiety is adeoxyribose sugar. A polyphosphate chain contains m phosphates, where mis an integer from three to eight. Three variations of the label portion(“L”) in structure I are shown in the structures II, III, and IV, andall of these comprise an unbranched alkyl group, an amine, and apolyproline chain of length X (“ProX”) that is bound to both theacceptor chromophore (“A”) and the donor chromophore (“D”). In certainpreferred embodiments, X is an integer of at least about 5 to 40, morepreferably 5 to 20, and its length in any particular compound dependentupon various experimental considerations, for example, the desired FRETefficiency and the characteristics and positions of the chromophoresbound thereto. In some embodiments, a polyproline chain of 6 to 14proline residues provides a preferred separation or distance betweenchromophores in a FRET label. Either the donor or acceptor chromophoremay attached to the polyproline chain at a position proximal to thenucleobase portion of the compound, as shown in the structures II andIII, respectively. Further, the polyproline chain may be attacheddirectly to the amine group, with the donor and acceptor chromophoresattached at positions along its length that promote a desired FRETefficiency, as shown in structure IV. In some preferred embodiments, theacceptor and donor chromophores are linked to the polyproline chain byamino- and carboxyl-terminal glycine and cysteine residues,respectively, and vice versa, by chemical methods known to those ofordinary skill in the art. See, e.g., Schuler, B. (2005) Proc. Natl.Acad. Sci. 102(8):2754-2759, incorporated by reference herein in itsentirety for all purposes. Further, although unbranched alkyl groups areshown in these exemplary embodiments, substituted alkyl groups may alsobe used in such linker structures, as described herein.

Although shown for purposes of illustration, it will be appreciated thatthe compounds of the invention encompass a range of variability,including, in particularly preferred aspects, that which is set forth inthe appended claims.

II. Analytical Reactions

The compounds and compositions of the invention have a variety ofdifferent uses and applications, including use in performing analyticalreactions. The labeled compounds of the invention are particularlyuseful in analytical reactions in which multiple components of areaction mixture must be differentially labeled. The labeled compoundsof the invention are also particularly useful in analytical reactions inwhich the number of preferred labeling moieties is limited by exclusionof labeling moieties that negatively impact the analytical reaction.

In certain aspects, the labeled compounds of the invention areparticularly useful in performing nucleic acid analyses. For example,such compounds may be used to detect association of nucleotide analogswith other reaction components, e.g., resulting in incorporation of thenucleotide analogs into a growing nucleic acid (e.g., DNA or RNA)strand. While preferred embodiments of the invention relate to nucleicacid molecules, including nucleotide analogs, oligonucleotides, andpolynucleotides that include the foregoing features, the principles ofthe invention are equally applicable to a broad range of reactants,labeling groups, and/or enzyme systems, including, e.g., kinases,phosphatases, ribosomes, receptors, helicases, ligands, nucleases,phosphatases, kinases, ligases, substrates, complexes, binding partners,etc. For ease of discussion however, the invention is described in termsof chromophore-labeled nucleotide analogs and their interaction withnucleic acid processing enzymes, including DNA and RNA polymerases,reverse transcriptases, nucleases, ligases, helicases, and the like,with DNA and RNA polymerases being particularly preferred enzymesystems. In addition to their use in sequencing, the analogs of theinvention are also equally useful in a variety of other genotypinganalyses, e.g., SNP genotyping that uses single-base extension methods,real-time monitoring of amplification, RT-PCR methods, and the like.

In particularly preferred embodiments, the labeled compounds of theinvention comprise chromophore-labeled nucleotide analogs used inenzymatic reactions, particularly real-time analytical reactions whereone observes chemical reactions through the detection of an emission oflight. For example, labeled nucleotide analogs are particularly usefulin polymerization reactions in which the label is excited during thesynthesis process by exposure to excitation illumination. Oneparticularly important example of such a reaction includespolymerase-mediated, template-dependent synthesis of nucleic acids thatcan be observed using real-time techniques for a variety of desiredgoals, including in particular determination of information about thetemplate sequence. A number of methods have been proposed fordetermination of sequence information using incorporation of fluorescentor fluorogenic nucleotides into the synthesized strand by a DNA or otherpolymerase, and the compositions and methods of the invention areapplicable to these methods. While several of these methods employiterative steps of nucleotide introduction, washing, opticalinterrogation, and label removal, certain preferred uses of thesecompositions utilize “real-time” determination of incorporation, inparticular during processive incorporation of nucleotides into a nascentstrand. Such methods are described in detail in, for example, U.S. Pat.Nos. 7,056,661, 7,052,847, 7,033,764 and 7,056,676, the full disclosuresof which are incorporated herein by reference in their entirety for allpurposes.

In certain embodiments, a reaction of interest, e.g., a polymerasereaction, can be isolated within an extremely small observation volumethat effectively results in observation of individual molecules, forexample, by using optical techniques that illuminate small volumesaround the complex with excitation radiation, e.g., total internalreflection microscopy (TIRF) methods, waveguide arrays, opticalconfinements like nanoholes and Zero Mode Waveguides (ZMWs), and thelike, and combinations thereof. In preferred embodiments, such methodsprovide optical resolvability between individual reaction sites andallow observation of a single reaction without interference from otherreactions occurring in the reaction mixture. For example, one canidentify individual nucleobase incorporation events based upon theoptical signature of the label portion of a labeled nucleotide analog ascompared to non-incorporated, randomly diffusing labeled nucleotideanalogs; or individual ligands binding to a receptor of interest; orindividual amino acids incorporated into a nascent polypeptide strand.In a preferred aspect, such small observation volumes are provided byimmobilizing the polymerase enzyme within an optical confinement, suchas a Zero Mode Waveguide (ZMW). For a description of ZMWs and othermeans of reducing observation volumes and providing opticalresolvability for monitoring individual molecules, enzymes, complexes,and the like, and their application in single molecule analyses, andparticularly nucleic acid sequencing, see, e.g., U.S. Ser. No.12/560,308, filed Sep. 15, 2009; Published U.S. Patent Application Nos.2003/0044781, 2008/0128627, 2008/0152281, and 2008/01552280; and U.S.Pat. Nos. 6,917,726, 7,013,054, 7,181,122, 7,292,742, 7,170,050, and7,302,146, each of which is incorporated herein by reference in itsentirety for all purposes.

In accordance with one aspect of the methods of invention, the compoundsdescribed herein are used in analyzing nucleic acid sequences using atemplate-dependent sequencing reaction to monitor the template-dependentincorporation of specific nucleotide analogs into a synthesized nucleicacid strand, and thereby determine a sequence of nucleotides present ina template nucleic acid strand. In certain specific embodiments, atemplate-dependent sequencing reaction comprises providing a templatenucleic acid (e.g., DNA or RNA) complexed with a polymerase enzyme in atemplate-dependent polymerization reaction, contacting the polymeraseand template nucleic acid with a labeled nucleotide analog of theinvention, detecting whether or not the labeled nucleotide analog isincorporated into a nascent nucleic acid strand during thepolymerization reaction, and identifying a base in the template strandbased upon incorporation of the labeled nucleotide analog. Suchanalytical reactions systems can comprise intramolecular FRET labels,intermolecular FRET labels (e.g., where one label is on or proximal tothe polymerase and the other label is on the nucleotide), non-FRETlabels, or a combination thereof. When a particular base in the templatestrand is encountered by the polymerase during the polymerizationreaction, it complexes with an available nucleotide analog that iscomplementary to the template nucleotide, and that analog isincorporated into the nascent and growing nucleic acid strand, e.g., bycleaving between the α and β phosphorus atoms in the analog, andconsequently releasing the labeling group (or a portion thereof). Theincorporation event is detected, either by virtue of a longer presenceof the labeled analog in the complex, or by virtue of release of thelabel group into the surrounding medium. By providing each differenttype of nucleotide analog with a distinguishable label, e.g., having adistinguishable emission spectrum, identification of the label of anincorporated analog allows identification of that analog, andconsequently the complementary sequence of the template can be deducedbased on the sequence of nucleotides incorporated into the nascentstrand. For example, different nucleotide analogs may comprise the sameFRET labeling moieties in different conformations, or different FRETlabels altogether, or non-FRET labels, or different acceptor labels thatemit different emission spectra in the presence of the same donor label,e.g., on the polymerase or at the reaction site, or a combinationthereof. For example, by changing the location of an acceptor group onthe nucleotide analog, the distance from a donor group, e.g., on thepolymerase, and the resulting emission spectrum is changed.Alternatively or additionally, the location of the donor group can alsobe adjusted to change the FRET efficiency of the FRET label. In someembodiments, all labeled nucleotide analogs in the reaction comprise aFRET label; in other embodiments, some of the labeled nucleotide analogsin the reaction comprise a FRET label and other labeled nucleotideanalogs in the reaction comprise a non-FRET label. In preferred aspects,labeled compounds of the invention present in the reaction are compoundsanalogous to at least one of the four natural nucleotides, A, T, G andC. In preferred embodiments, incorporation of labeled nucleotide analogsis detected in real-time during nascent strand synthesis. Preferably,the foregoing process is carried out so as to permitdetection/identification of individual nucleotide incorporation events,through the use of, for example, an optical confinement that allowsobservation of an individual polymerase enzyme, or through the use of aheterogeneous assay system, where label groups released fromincorporated analogs are detected. In particularly preferred aspects,the polymerase enzyme/template complex is provided immobilized within anoptical confinement that permits observation of an individual complex,e.g., a zero mode waveguide.

In accordance with one aspect of the methods of invention, the compoundsdescribed herein are used in analyzing polypeptide sequences using anmRNA translation reaction to monitor and determine the incorporation ofamino acids into a synthesized polypeptide chain. In certain specificembodiments, a translation reaction comprises providing a template mRNAcomplexed with a ribosome, contacting the ribosome and template mRNAwith a labeled tRNA analog, detecting whether or not the amino acidcarried by the labeled tRNA analog is incorporated into a nascentpolypeptide chain, and identifying an amino acid so incorporated, e.g.,based upon the label carried by the tRNA analog. For example, theincorporation event can be detected by virtue of a longer presence ofthe labeled tRNA analog in the complex. By providing each different typeof tRNA analog with a distinguishable label, e.g., having adistinguishable emission spectrum, identification of the label of a tRNAanalog that associates with the ribosome allows identification of theanalog and the amino acid it carries. Such analytical reactions systemscan comprise intramolecular FRET labels, intermolecular FRET labels(e.g., where one label is on or proximal to the ribosome and the otherlabel is on the tRNA), non-FRET labels, or a combination thereof. Forexample, different tRNA analogs may comprise the same FRET labelingmoieties in different conformations, or different FRET labelsaltogether, or non-FRET labels, or different acceptor labels that emitdifferent emission spectra in the presence of the same donor label,e.g., on the ribosome or at the reaction site, or a combination thereof.For example, by changing the location of an acceptor group on the tRNAanalog, the distance from a donor group, e.g., on the ribosome, and theresulting emission spectrum is changed. Alternatively or additionally,the location of the donor group can also be adjusted to change the FRETefficiency of the FRET label. Various labeling strategies for and otheraspects of translation-based protein sequencing are provided in U.S.Ser. No. 61/186,645, filed Jun. 12, 2009, the disclosure of which isincorporated herein by reference in its entirety for all purposes. Insome embodiments, all labeled tRNA analogs in the reaction comprise aFRET label; in other embodiments, some of the labeled tRNA analogs inthe reaction comprise a FRET label and other labeled tRNA analogs in thereaction comprise a non-FRET label. In preferred embodiments,incorporation of amino acids is detected in real-time duringtranslation. Preferably, the foregoing process is carried out so as topermit detection/identification of individual amino acid incorporationevents, through the use of, for example, an optical confinement thatallows observation of an individual ribosome. In particularly preferredaspects, the ribosome/mRNA complex is provided immobilized within anoptical confinement that permits observation of an individual complex,e.g., a zero mode waveguide.

As noted above, in a typical template-dependent synthesis reaction, eachnucleotide to be incorporated bears a label that identifies thenitrogenous base portion of the nucleotide, so in typical nucleic acidsynthesis reactions four such labels are present to differentially labelthe four different nucleotides (e.g., A, C, G, and T). Detection ofmultiple chromophores in a single analytical reaction introduceschallenges related not only to excitation and detection, but also thevariations in performance and the potential for negative effectsassociated with the use of certain chromophores (e.g., photodamage). Incertain aspects, the compounds, methods, and compositions providedherein are particularly useful for reducing the number of chromophoresneeded to differentially label multiple components of an analyticalreaction. In certain embodiments, this is accomplished by using a singlecombination of FRET label moieties to label multiple reaction components(“labeled compounds”), and varying the structure of the labeledcompounds such that different reactive portions (e.g., nucleotideanalogs) are linked to FRET labels having different orientations andtherefore producing different and distinct emission spectra. In certainaspects, a single combination of FRET label moieties may be used toprovide at least about 2-10 different emission spectra based on theorientation of the chromophores relative to one another, and can be usedto label compounds including not only nucleotide analogs, but also othertypes of reactants described herein and elsewhere, including enzymes,tRNAs, binding partners, ligands, receptors, cofactors, and the like.Further, as described elsewhere herein, such FRET labels can beintramolecular FRET labels or intermolecular FRET labels, e.g., in whicha donor chromophore is attached to one labeled compound and an acceptorchromophore is attached to a second labeled compound. In certainembodiments, multiple different reactants carry the same acceptorchromophore, and the configuration of the acceptor chromophore on eachreactant is designed to produce a distinct and recognizable emissionspectrum that can be used to distinguish between the different reactantswhen they undergo FRET with the donor chromophore.

In certain embodiments, FRET-labeled compounds may be combined withnon-FRET-labeled compounds in a single analytical reaction. For example,a single reaction may include at least one or two of the nucleotideanalogs that comprise a FRET label and at least one or two nucleotideanalogs that do not comprise a FRET label, with identification of theincorporated nucleotide analog based at least in part on a comparison ofthe emission spectra generated during polymerization of the nascentstrand. In preferred embodiments, incorporation of labeled nucleotideanalogs, whether FRET-labeled or non-FRET-labeled (i.e., comprising adetectable label that does not undergo FRET), is detected in real-timeduring nascent strand synthesis. In a specific embodiment of apolymerase-mediated, template-directed synthesis reaction, twochromophores may be used to distinctly label the four nucleotide analogsto be incorporated into the nascent strand. For example, a firstnucleotide analog is labeled with chromophore A, a second nucleotideanalog is labeled with chromophore B, a third nucleotide analog islabeled with the A-B FRET pair in a first orientation, and a fourthnucleotide analog is labeled with the A-B FRET pair in a secondorientation. In this way, four different emission spectra are producedin a single analytical reaction using only two chromophores, two of theemission spectra being produced from FRET pairs having differentconformations and therefore different FRET efficiencies. Although thisprophetic example describes a reaction comprising two FRET-labeledcompounds that comprise the same two chromophores, the present inventionalso contemplates reactions in which multiple FRET-labeled compounds ina single reaction may share more or fewer than two chromophores.Further, as noted above, reactions utilizing the labeled compounds ofthe invention may comprise a combination of FRET and non-FRET labeledcompounds, may comprise intramolecular and/or intermolecular FRETlabels, and may comprise labeled compounds having multiple labelingmoieties that do not undergo FRET.

In various embodiments of the methods provided herein, one or morecomponents of an analytical reaction are immobilized. Suchimmobilization can be engineered in various ways using methods wellknown to the ordinary practitioner and routinely practiced in the art.For example, immobilization of enzymes or other proteins may employ anyof a variety of techniques, including, for example, in vivobiotinylation of a N- or C-terminal peptide tag on the protein (e.g.AviTag (Avidity)) (see, e.g., D. Beckett, et al., Protein Sci 1999, 8,921, which is incorporated herein by reference in its entirety for allpurposes), which provides high efficiency of biotinylation andpreservation of enzymatic activities or other characteristics, such asbinding specificity, higher order structure, etc. A variety of othersurface treatments are also optionally exploited to avoid non-specificinteractions of free reagents and the surfaces of the illuminationvolume, which could give rise to aberrant signals. For example,polyphosphonate and silane-based surface coatings may be exploited thatmediate enzyme attachment to the transparent floor of a zero modewaveguide while blocking non-specific attachments to the metal top andside wall surfaces (see, e.g., J. Eid, et al. (incorporated hereinabove) and J. Korlach, et al., Proc Natl Acad Sci USA 2008, 105, 1176,which is incorporated herein by reference in its entirety for allpurposes).

One skilled in the art will appreciate that there are many ways ofimmobilizing nucleic acids and proteins into an optical confinement,whether covalently or non-covalently, via a linker moiety, or tetheringthem to an immobilized moiety. These methods are well known in the fieldof solid phase synthesis and micro-arrays (Beier et al., Nucleic AcidsRes. 27:1970-1-977 (1999)). Non-limiting exemplary binding moieties forattaching either nucleic acids or polymerases to a solid support includestreptavidin or avidin/biotin linkages, carbamate linkages, esterlinkages, amide, thiolester, (N)-functionalized thiourea, functionalizedmaleimide, amino, disulfide, amide, hydrazone linkages, among others.

In some embodiments, antibodies specific for one or more reactioncomponents are used to bind and immobilize the reaction components toreaction sites, e.g., particular locations on a substrate, in a way thatmaintains their ability to participate in an analytical reaction ofinterest. This method of immobilization is especially useful where thereactants are being collected from a sample to be applied to thereaction site. Further, nucleic acid molecules may be directly linked toa reaction site, or may be indirectly linked, e.g., through interactionwith a primer or other moiety directly linked to the reaction site. Sucha primer may be designed to be complementary to a particular region ormultiple regions of interest in the RNA template(s), may be randomlygenerated, or may be an olito(dT) that will anneal to the poly-dA tailon mRNAs. In addition, a silyl moiety can be attached to a nucleic aciddirectly to a substrate such as glass using methods known in the art. Incertain embodiments, a biomolecular complex is assembled in at areaction site, e.g., by first immobilizing an enzyme component. In otherembodiments, such a complex is assembled in solution prior toimmobilization. Various additional methods for immobilizing molecularcomplexes are provided, e.g., in U.S. Pat. No. 7,476,503, which isincorporated herein by reference in its entirety for all purposes. Inpreferred embodiments, reaction components are immobilized at a reactionsite such that signals emitted from each resulting analytical reactionare optically resolvable from signals emitted from every otheranalytical reaction at every other reaction site, e.g., on a substrate.Immobilized reaction component may or may not comprise a detectablelabel, e.g., one or more chromophores of a FRET label.

Where desired, an enzyme or other protein reaction component to beimmobilized may be modified to contain one or more epitopes such as Myc,HA (derived from influenza virus hemagglutinin), poly-histadines, and/orFLAG, for which specific antibodies are available commercially. Inaddition, proteins can be modified to contain heterologous domains suchas glutathione S-transferase (GST), maltose-binding protein (MBP),specific binding peptide regions (see e.g., U.S. Pat. Nos. 5,723,584,5,874,239 and 5,932,433), or the Fc portion of an immunoglobulin. Therespective binding agents for these domains, namely glutathione,maltose, and antibodies directed to the Fc portion of an immunoglobulin,are available and can be used to coat the surface of an opticalconfinement of the present invention.

The binding moieties or agents of the reaction components theyimmobilize can be applied to a reaction site by conventional chemicaltechniques which are well known in the art. In general, these procedurescan involve standard chemical surface modifications of a support,incubation of the support at different temperature levels in differentmedia comprising the binding moieties or agents, and possible subsequentsteps of washing and cleaning. Further, various means of loadingmultiple biological reactions onto a substrate are known to those ofordinary skill in the art and are described further, e.g., in U.S. Ser.No. 61/072,641, incorporated herein by reference in its entirety for allpurposes.

III. Systems

The present invention also employs the nucleotide analog compounds andcompositions described herein in conjunction with overall analyticalsystems. Typically, such systems employ a reaction region or reactionsite that is typically disposed in a reaction vessel or well. By way ofexample, such systems may include a substrate component upon which areimmobilized, e.g., a polymerase/template/primer complex, for use in thedetermination of nucleic acid sequence information of the template,which may be derived from an organism of interest.

Because the compositions of the invention are preferablychromophore-labeled, it will be appreciated that the preferred systemsof the invention will comprise chromophore emission detectionfunctionalities. Examples of such systems include those described in,e.g., Published U.S. Patent Application Nos. 2007/0036511 and2007/095119 and U.S. patent application Ser. No. 11/901,273 filed Sep.14, 2007, the full disclosures of which are incorporated herein byreference in their entirety for all purposes. One such system isschematically illustrated in FIG. 6 .

As shown in FIG. 3 , the system 300 includes a substrate 302 thatincludes a plurality of discrete sources of chromophore emissionsignals, e.g., an array of zero mode waveguides 304. An excitationillumination source, e.g., laser 306, is provided in the system and ispositioned to direct excitation radiation at the various signal sources.This is typically done by directing excitation radiation at or throughappropriate optical components, e.g., dichroic 108 and objective lens310, that direct the excitation radiation at the substrate 302, andparticularly the signal sources 304. Emitted signals from the sources304 are then collected by the optical components, e.g., objective 310,and passed through additional optical elements, e.g., dichroic 308,prism 312 and lens 314, until they are directed to and impinge upon anoptical detection system, e.g., detector array 316. The signals are thendetected by detector array 316, and the data from that detection istransmitted to an appropriate data processing unit, e.g., computer 318,where the data is subjected to interpretation, analysis, and ultimatelypresented in a user ready format, e.g., on display 320, or printout 322,from printer 324. As will be appreciated, a variety of modifications maybe made to such systems, including, for example, the use of multiplexingcomponents to direct multiple discrete beams at different locations onthe substrate, the use of spatial filter components, such as confocalmasks, to filter out-of focus components, beam shaping elements tomodify the spot configuration incident upon the substrates, and the like(See, e.g., Published U.S. Patent Application Nos. 2007/0036511 and2007/095119, and U.S. patent application Ser. No. 11/901,273, all ofwhich are incorporated herein by reference in their entireties for allpurposes).

In certain aspects, the methods provide a means for studying analyticalreactions in vitro by immobilizing at least one component of aanalytical reaction in an optical confinement, labeling at least oneother component, and detecting signals from the optical confinementduring the reaction in real time. An optical confinement ispreferentially configured to provide tight optical confinement so only asmall volume of the reaction mixture is observable, i.e., signals canonly be detected from a small volume of the reaction mixture. In certainembodiments, optical confinement technologies include zero modewaveguides, total internal reflection microscopy (TIRF), and/or opticalwaveguides (planar or otherwise configured). For example, in embodimentsin which excitation illumination is used to excitechromophore-containing labels, the tight optical confinement allows onlya small volume of the reaction mixture to be illuminated, and thereforelimits excitation to only those chromophores within that small volume.As such, only the chromophores present in the small illuminated volumeare excited and emit signals that are detectable by the optical system.This feature of the invention is useful for reducing the backgroundsignal from freely diffusing detectably labeled components in thereaction mixture, thereby enabling the use of physiologicalconcentrations of these reagents. Some such optical confinements andmethods of manufacture and use thereof are described at length in, e.g.,U.S. Pat. Nos. 7,302,146, 7,476,503, 7,313,308, 7,315,019, 7,170,050,6,917,726, 7,013,054, 7,181,122, and 7,292,742; U.S. Patent PublicationNos. 20080128627, 20080152281, and 200801552280; and U.S. Ser. No.11/981,740, all of which are incorporated herein by reference in theirentireties for all purposes. The optical confinements can be furthertailored in various ways for optimal confinement of an analyticalreaction of interest. In particular, the size, shape, and composition ofthe optical confinement can be specifically designed for containment ofa given enzyme complex and for the particular label and illuminationscheme used.

Providing such individually resolvable configurations can beaccomplished through a number of mechanisms, and typically involvesimmobilization of at least one component of an analytical reaction at areaction site. For example, by providing a dilute solution of complexeson a substrate surface suited for immobilization, one will be able toprovide individually optically resolvable complexes. (See, e.g.,European Patent No.1105529 to Balasubramanian, et al., the fulldisclosure of which is incorporated herein by reference in its entiretyfor all purposes.) Alternatively, one may provide a low densityactivated surface to which complexes are coupled. (See, e.g., PublishedInternational Patent Application No. WO 2007/041394, the full disclosureof which is incorporated herein by reference in its entirety for allpurposes). Such individual complexes may be provided on planarsubstrates or otherwise incorporated into other structures, e.g., zeromode waveguides or waveguide arrays, to facilitate their observation. Inpreferred embodiments, a substrate comprises at least one opticalconfinement in which a molecule or molecular complex is immobilized andmonitored. The optical confinement is a structure configured to isolatethe immobilized molecule/complex from any other molecule/compleximmobilized on the substrate, and in particular to isolate anydetectable signals emitted from the optical confinement from any othersignals emitted from any other optical confinements on the substrate.Such isolation allows the practitioner of the instant invention tounambiguously assign a detected signal to a single optical confinementon the substrate, and therefore to a single analytical reaction on thesubstrate.

IV. Kits

The present invention also provides kits useful for exploiting thecompounds and compositions described herein in a number of applications.In a first respect, such kits typically include one or more labeledcompounds (e.g., labeled compounds that undergo intramolecular orintermolecular FRET, e.g., FRET-labeled nucleotide analogs) of theinvention packaged in a fashion to enable their use, and preferably acomposition comprising a set of differentially labeled compounds. Incertain embodiments, a subset of the set of differentially labeledcompounds comprise FRET labels and a subset of the differentiallylabeled compounds comprise non-FRET labels, e.g., comprising a single ormultiple labeling moieties. In certain embodiments, a kit comprises atleast four different nucleotide analogs, namely those that are analogousto A, T, G, and C, where each bears a detectably different labelinggroup to permit its individual identification in the presence of theothers. In other embodiments, a kit comprises different tRNA analogsthat are analogous to natural tRNA molecules, at least some of whichbear detectably distinct labeling groups to permit their individualidentification in polypeptide synthesis reactions. Other kits compriselabeled components for other types of analytical reactions, includingantibody assays, hybridization assays, genotyping assays, enzymaticassays, binding assays, etc. Depending upon the desired application, thekits of the invention optionally include additional reagents which mayor may not comprise labels, such as enzymes for performing analyticalreactions employing the labeled compounds of the invention, and otherreagents, such as buffer solutions and/or salt solutions, including,e.g., divalent metal ions, i.e., Mg⁺⁺, Mn⁺⁺ and/or Fe⁺⁺, cofactors,standard solutions, e.g., dye standards for detector calibration. Insome preferred embodiments, a kit of the invention includes polymeraseenzymes for performing template-dependent synthesis employing thelabeled nucleotide analogs of the invention, ribosomes for performingprotein synthesis reactions, transcription factors for performingnucleic acid binding assays, etc. Each of the different types of labeledcompounds in a kit will typically comprise a distinguishable labelinggroup, as set forth above. Examples of polymerases that are preferablyincluded in a kit of the invention include, e.g., phi29 derivedpolymerases and the polymerase enzymes described in, e.g., PublishedInternational Patent Application Nos. WO 2007/075987, WO 2007/075873,and WO 2007/076057, the full disclosures of which are incorporatedherein by reference in their entirety for all purposes. The polymerasemay comprise a label (e.g., a donor) that undergoes FRET with a label(e.g., an acceptor) attached to one or more of the nucleotide analogs,e.g., where a given analog having a first nucleobase is configured suchthat the emission spectrum during interaction with the polymerase isdetectably distinct from the interaction of any other analog having anyother nucleobase. Kits for performing template-directed synthesisreactions may optionally include primer and/or template sequences, aswell. Such kits also typically include instructions for use of thecompounds and other reagents in accordance with the desired applicationmethods, e.g., nucleic acid sequencing, and the like.

In addition, in particularly preferred aspects, the kits of theinvention will typically include a reaction substrate that includesreaction regions for carrying out and observing the analyticalreactions, e.g., synthesis reactions for identification of sequenceinformation. Such substrates include, e.g., multi-well micro or nanoplates, as well as arrayed substrates, e.g., planar transparent arraysthat include discrete reaction regions defined by, e.g., structural,chemical or other means. For example, patterned arrays of complexes maybe provided disposed upon planar transparent substrates for observation.Preferably, such substrates provide optical resolvability betweenindividual reactions, e.g., immobilized at different reaction sites onthe substrate. Alternatively and preferably, the substrate componentcomprises an array or arrays of optically confined structures like zeromode waveguides and/or optical waveguide arrays. Examples of arrays ofzero mode waveguides are described in, e.g., U.S. Pat. No. 7,170,050,the full disclosure of which is incorporated herein by reference in itsentirety for all purposes. Examples of optical waveguide arrays aredescribed, e.g., in U.S. Ser. No. 12/560,308, filed Sep. 15, 2009, andU.S. Patent Publication Nos. 2008/0128627, 2008/0152281, and2008/01552280.

V. Exemplary Compounds and Methods for Synthesis

Although the exemplary compounds and synthesis schemes provided beloware focused on FRET dyes that comprise cyanine dyes, in particular Cy3and Cy5, the following examples are simply exemplary embodiments ofcompounds and methods of making such compounds for use in thecompositions and methods of the invention. As such, it will beunderstood that other dyes may also be substituted in these exemplarycompounds and synthesis schemes provided.

FIG. 4 provides structures for three FRET dyes composed of Cy3 and Cy5fluorescent dye, each of which has an inter-dye linker of a differentlength. By adding extra aminomethyl benzoic acid (amb) groups betweenthe donor dye (Cy3) and the acceptor dye (Cy5) we can vary the distancebetween the pair and thus control the energy efficiency as discussedabove.

The compounds of the invention are generally synthesizable using methodsknown to those of ordinary skill in the art. For example, synthesis ofthe first structure illustrated in FIG. 4 (“Cy5-Cy3”) was carried outand the synthesis scheme is shown in FIG. 5 . Specifically, nitrationand amidomethylation of 2,3,3-trimethylindolenine (1) with nitric acidand sulfuric acid was followed by reaction withN-(hydroxymethyl)phthalimide gives the phthalimide (2), which was heatedwith propanesultone to form the quarternary amine (3). Reaction of (3)with N-(5-carboxypentyl)-2,3,3-trimethyl-5-sulfo-indolenine andN,N′-diphenylformamidine gave the carbocyanine compound (4).Deprotection of the phthalimide group with concentrated hydrochloricacid gave the desired bifunctional cyanine dye (5). Coupling of thebifunctional cyanine dye with Cy5-mono NHS ester (6) gave the Cy5-Cy3FRET dye (7).

Synthesis of the second structure illustrated in FIG. 4 (“Cy5-amb-Cy3”)is shown in FIG. 6 , and synthesis of the third structure illustrated inFIG. 4 (“Cy5-amb2-Cy3”) is shown in FIG. 7 . The Cy5-amb-Cy3 labelcomprises an aminomethyl benzoic acid linking the two dye molecules; andthe Cy5-amb2-Cy3 label comprises two aminomethyl benzoic acid groupslinking the two dye molecules. Reaction of the amino carboxylic acidbifunctional cyanine compound (5) with the trifluoroacetate (TFA)protected aminomethylbenzoic NHS ester (8) in N,N-dimethylformamidefollowed by hydrolysis with ammonium hydroxide gives the Cy3-amb-NH2(9). Coupling of Cy3-amb-NH2 (9) with Cy5-mono NHS ester (6) gives theCy5-amb-Cy3-FRET dye (10). Similarly, the Cy5-amb2-Cy3 FRET dye (12) canthen be prepared starting with Cy3-amb-NH2 (9) following the same stepsas described above.

FIG. 8 provides a general synthetic strategy for the preparation ofCy5-Linker-Cy3 FRET dyes following the same synthetic approaches asdescribed above for the preparation of Cy5-amb2-Cy3 (12). Reaction ofthe amino carboxylic acid bifunctional cyanine compound (5) with a TFAprotected linker-1 NHS ester followed by hydrolysis with ammoniumhydroxide gives the Cy3-L1-NH2 (13). Coupling of the Cy3-L1-NH2 (13)with a second TFA protected linker-2 NHS ester and again followed byhydrolysis with ammonium hydroxide gives the double linker cyanine dye,Cy3-L1-L2-NH2 (14), which then reacts with Cy5-mono-NHS ester to givethe desired Cy5-L2-L1-Cy3 FRET dye (15). The same procedure can also beapplied for the synthesis of a Cy5-multiple linkers-Cy3 FRET dyes.

FIG. 9 illustrates one embodiment of a synthetic scheme for constructionof FRET-labeled nucleotide analogs comprising a hexaphosphate chain.Transformation of the Cy5-linkers-Cy3-COOH (15) to the corresponding NHSester can be achieved with various activation methods, such as theclassical CDI/NHS, TSTU, EDAC/NHS or DCC/NHS. The resultant NHS ester(16) is then coupled to the aminohexaphosphate deoxy nucleotide to givethe corresponding FRET-labeled nucleotide analog (17).

FIG. 10 depicts an example of using polyproproline as the rigid linker,and the synthesis of Cy5-pro6-Cy3-dG6P is demonstrated. Reaction ofaminohexaphosphate deoxyguanosine, dG6P-NH2 (18), with TFA protected Cy3NHS ester (19) followed by hydrolysis with ammonium hydroxide gives thecompound dG6P-Cy3-NH2 (20). Reaction of dG6P-Cy3-NH2 (20) withiodoacetic acid N-hydroxysuccinimide ester (21) gives the correspondingadduct, compound (22), which then reacts with the amino hexaprolinethiol (23) to give the coupling product (24). Reaction of compound (24)with Cy5-mono NHS ester gives the FRET-labeled nucleotide analog (25)which has a rigid hexaproline linker between the donor Cy3 dye and theacceptor Cy5 dye.

FIG. 11 provides emission spectra for Cy5 and Cy3 dyes. FIG. 12 providesan emission spectrum for a Cy5-Cy3 FRET dye exhibiting an energytransfer of greater than 50%. The FRET transfer from Cy3 to Cy5 changesthe ratio of the intensities of Cy3 to Cy5 emissions such that theintensity of the Cy3 emission is approximately one-fourth that of theCy5 emission. FIG. 13 depicts a computer-simulated emission spectrum ofa compound Cy5-X-Cy3 that shows a 50% energy transfer that provides fora ratio of intensities of Cy3 to Cy5 emissions that is approximately1:1. FIG. 14 depicts a computer-simulated emission spectra of a compoundCy5-Y-Cy3 that shows a less than 50% energy transfer, which changes theratio of the intensities of Cy3 to Cy5 emissions such that the intensityof the Cy3 emission is approximately four-fold greater that of the Cy5emission. As described herein, these unique fluorescent emission spectracan be used as markers for different labeled compounds in analyticalreactions. For example, in some preferred embodiments, such labeledcompounds are labeled nucleotides in sequencing-by-synthesis analyticalreactions using only one laser source to provide emission spectra thatidentify the nucleobase in a given labeled nucleotide incorporated intoa nascent nucleic acid strand.

Although described in some detail for purposes of illustration, it willbe readily appreciated that a number of variations known or appreciatedby those of skill in the art may be practiced within the scope ofpresent invention. For example, although some of the exemplary FRETlabels and labeled compounds described in the specification and figurescomprise Cy3 and Cy5 fluorescent dyes, the compounds, compositions, andmethods are not limited to the cyanine dyes and other groups offluorescent dyes such as rhodamine derivatives (e.g., Alexa dyes(Molecular Probes/Invitrogen/Life Technologies) and Dylight dyes(Dyomics/Thermo Fisher Scientific)), and other labeling moieties knownin the art (e.g., quantum dots) can be selected as candidates for eitherthe donor dyes or the acceptor dyes. While various embodiments hereinare discussed in terms of their application to single moleculesequencing, e.g., using ZMWs, it will be appreciated that the methodsand systems are also applicable for use with monitoring of otherenzymatic systems, e.g., immunoassays, drug screening, and the like,and/or in non-confined detection systems, e.g., systems that do not useZMWs or similar confinement schemes. Further, although some of theembodiments are described in terms of specific types of reactioncomponents (e.g., nucleotide analogs, tRNA analogs, and the like), theinvention is not limited to use with these exemplary reaction componentsand the various types of intramolecular and intermolecular FRET labelsprovided herein can be used on countless other types of reactioncomponents known to those of ordinary skill in the art. All terms usedherein are intended to have their ordinary meaning unless an alternativedefinition is expressly provided or is clear from the context usedtherein. To the extent any definition is expressly stated in a patent orpublication that is incorporated herein by reference, such definition isexpressly disclaimed to the extent that it is in conflict with theordinary meaning of such terms, unless such definition is specificallyand expressly incorporated herein, or it is clear from the context thatsuch definition was intended herein. Unless otherwise clear from thecontext or expressly stated, any concentration values provided hereinare generally given in terms of admixture values or percentages withoutregard to any conversion that occurs upon or following addition of theparticular component of the mixture. To the extent not already expresslyincorporated herein, all published references and patent documentsreferred to in this disclosure are incorporated herein by reference intheir entireties for all purposes.

What is claimed is:
 1. A reaction mixture for nucleic acid sequencing,comprising: a first nucleotide analog and a second nucleotide analog,wherein the first nucleotide analog comprises a first fluorescent labelthat produces a first signal in response to excitation illumination, andthe second nucleotide analog comprises a second fluorescent label thatproduces a second signal in response to excitation illumination, whereinthe first signal and second signal comprise peaks at the samewavelengths, and wherein the first signal and second signal havedistinct signal intensities, a template nucleic acid, and a polymeraseenzyme, wherein the polymerase and template comprise apolymerase/template complex.
 2. The reaction mixture of claim 1, whereinat least one of the first and second signals comprises distinct emissionsignal intensities at a plurality of different wavelengths.
 3. Thereaction mixture of claim 1, wherein the first fluorescent labelcomprises a first FRET label having a first FRET efficiency, and thesecond fluorescent label comprises a second FRET label having a secondFRET efficiency different from the first FRET efficiency, wherein thefirst FRET label and the second FRET label each comprise at least twochromophores that engage in FRET.
 4. The reaction mixture of claim 1,wherein the first and second FRET efficiencies are from about 0 to about100% of a maximum FRET efficiency for each of the first and second FRETlabels.
 5. The reaction mixture of claim 1, wherein the reaction mixturefurther comprises at least a third nucleotide analog, the thirdnucleotide analog comprising a third fluorescent label producing a thirdsignal in response to excitation illumination, the third signaldiffering from each of the first and second signals by at least one itsemission intensity.
 6. The reaction mixture of claim 1, wherein thereaction mixture further comprises at least a fourth nucleotide analog,the fourth reactant comprising a fourth fluorescent label producing afourth signal in response to excitation illumination, the fourth signaldiffering from each of the first, second and third signals by itsemission intensity.
 7. The reaction mixture of claim 1, wherein thereaction mixture further comprises at least four different nucleotideanalogs that are analogous to A, T, G, and C, where each bears adetectably different labeling group to permit its individualidentification in the presence of the others.
 8. The reaction mixture ofclaim 1, wherein the reaction mixture further comprises two additionalnucleotide analogs, wherein at least one of the first, second, andadditional nucleotide analogs comprises a FRET label and one othercomprises a non-FRET label.
 9. The reaction mixture of claim 1, whereinthe first and second nucleotide analogs comprise FRET labels having thesame constituent FRET label moieties in different orientations.
 10. Thereaction mixture of claim 1, wherein the reaction mixture furthercomprises nucleotide analogs having multiple labeling moieties that donot undergo FRET.
 11. The reaction mixture of claim 1, wherein thepolymerase is a phi29 polymerases.
 12. The reaction mixture of claim 1,wherein the reaction mixture further comprises a sequencing primer. 13.The reaction mixture of claim 1, wherein the reaction mixture furthercomprises one or more solutions for performing a sequencing reaction.14. The reaction mixture of claim 1, wherein the reaction mixturefurther comprises dye standards for detector calibration.
 15. Thereaction mixture of claim 1, wherein the polymerase/template complex isimmobilized.
 16. The reaction mixture of claim 1, wherein thepolymerase/template complex is immobilized within an opticalconfinement.
 17. The method of claim 1, wherein the fluorescent dyes areCy3, Cy3.5, Cy5, Cy5.5 derivatives or equivalents thereof.